Alpha-amylase variants

ABSTRACT

A variant polypeptide having alpha-amylase activity is disclosed. The variant polypeptide has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises at least one substitution of an amino acid residue with reference to SEQ ID NO: 2. The variant polypeptide has one or more altered properties as compared with a reference polypeptide having alpha-amylase activity. Such a variant polypeptide may be used in the preparation of a baked product.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to the European EP13157398.2 and alsothe U.S. provisional patent application 61/771,676, both filed on 1 Mar.2013, the contents of which are both incorporated herein by reference intheir entireties.

BACKGROUND

1. Field of the Invention

The invention relates to a variant polypeptide having alpha-amylaseactivity. The invention also relates to a nucleic acid sequence encodingsuch a variant, to a recombinant expression vector a said nucleic acidconstruct and to a recombinant host cell comprising a said expressionvector. Further, the invention relates to a method for producing analpha amylase via use of such a host cell. Also, the invention relatesto a method of producing an alpha-amylase polypeptide variant. Theinvention further relates to a composition comprising an alpha-amylasevariant, to use of such an alpha-amylase variant or alpha-amylasevariant-containing composition in the preparation of a baked product, toa process for the production of a baked product and to the resultingbaked product.

2. Description of Related Art

In bread making starch plays a major role in the crumb formation and therate of crumb staling of the baked bread. In dough starch is present asgranules, absorbing only a small amount of water. During baking thestarch gelatinization process is taking place. Amylose is leaking out ofthe granule and forms a continuous gel in the baking dough. Alreadyduring baking part of the amylose is re-crystallizing, resulting instiffening of the gel and setting of the crumb. At the same time wateris entering the granule and hydrating the amylopectin resulting inswelling of the granule. During storage of the bread over several days,the amylopectin starts to re-crystallize (also called retrogradation).The staling of bread is believed to be a direct reflection of theretrogradation of amylopectin. The starch and thus the breadcrumb becomemore rigid

The firmness of bread after a certain storage time is depending on theinitial softness, which is the softness after cooling down, and the rateof increase of firmness, the rate of staling.

Studies on bread staling have indicated that the starch fraction inbread recrystallizes during storage, thus causing an increase in crumbfirmness, which may be measured as an increase in hardness of breadslices.

The present invention relates to an alpha-amylase. Alpha-amylases havebeen used in industry for a long time.

Alpha-amylases have traditionally been provided through the inclusion ofmalted wheat or barley flour and give several advantages to the baker.Alpha-amylase is used to give satisfactory gas production and gasretention during dough leavening and to give satisfactory crust color.This means that if this enzyme is not used in sufficient amount, thevolume, texture, and appearance of the loaf are substantially impaired.Alpha-amylase occurs naturally within the wheat crop itself, measuredroutinely by Hagberg Falling Number (ICC method 107), and steps aretaken to minimise such variations by the addition of alpha-amylase atthe mill and through the use of specialty ingredients at the bakery asthe enzyme is of such critical importance.

In more recent times, alpha-amylase from cereal has been largelyreplaced with enzymes from microbial sources, including fungal andbacterial sources. Through use of biotechnology in strain selection,fermentation and processing, enzymes can be prepared from such microbialsources and this brings advantage over malt flour because the enzyme isof more controlled quality, relatively pure and more cost effective inuse.

The properties of alpha-amylases, and their technological effects, dohowever show important differences. Besides giving influence to gasproduction, gas retention and crust color, alpha-amylase can havebearing on the shelf-life of the baked product.

Starch within the wheat flour contains two principal fractions, amyloseand amylopectin, and these are organised in the form of starch granules.A proportion of these granules from hard-milling wheat varieties become“damaged”, with granules splitting apart as a consequence of the energyof milling. In the process of baking, the starch granules gelatinise;this process involves a swelling of the granule by the uptake of waterand a loss of the crystalline nature of the granule; in particularamylopectins within the native granule are known to exist ascrystallites and these molecules dissociate and lose crystallinityduring gelatinisation. Once the bread has been baked, amylopectinrecrystallises slowly over a numbers of days and it is thisrecrystallisation, or retrogradation of starch, that is regarded asbeing the principal cause of bread staling.

These varying forms of the starch and their interaction withalpha-amylase dictate the role the enzyme has with respect to bakingtechnology. Alpha-amylase from fungal sources, most typically comingfrom Aspergillus species, acts principally on damaged starch during themixing of dough and throughout fermentation/proof. The low heatstability of the enzyme means that the enzyme is inactivated duringbaking and, critically before starch gelatinisation has taken place,such that there is little or no breakdown of the starch from theundamaged fraction. As a consequence, fungal amylase is useful inproviding sugars for fermentation and color, but has practically novalue in extending shelf-life. Bacterial alpha-amylase, most typicallyfrom Bacillus amyloliquifaciens, on the other hand does bring extendedtemperature stability and activity during the baking of bread and whilestarch is undergoing gelatinisation. Bacterial amylase then leads tomore extensive modification of the starch and, in turn, the qualities ofthe baked bread; in particular the crumb of the baked bread can beperceptibly softer throughout shelf-life and can permit the shelf-lifeto be increased. However, while bacterial alpha-amylase can be usefulwith regard to shelf-life extension, it is difficult to use practicallyas small over-doses lead to an unacceptable crumb structure of large andopen pores, while the texture can become too soft and “gummy”.

There is a need for an alpha-amylase with improved performance inindustry, especially in the baking industry.

U.S. Pat. No. 4,598,048 describes the preparation of a maltogenicamylase enzyme. U.S. Pat. No. 4,604,355 describes a maltogenic amylaseenzyme, preparation and use thereof. U.S. RE38,507 describes anantistaling process and agent.

-   WO99/43793 discloses amylolytic enzyme variants.-   WO99/43794 maltogenic alpha-amylase variants.-   WO2004/081171 discloses and enzyme.-   WO2006/012899 discloses maltogenic alpha-amylase variants.

SUMMARY

The invention relates to variant polypeptides having alpha-amylaseactivity, i.e. to alpha-amylase variants. An alpha-amylase variant ofthe invention may have one or more improved properties in comparisonwith a reference polypeptide, the reference polypeptide typically havingalpha-amylase activity. A reference polypeptide may be a wild-typealpha-amylase, such as wild-type alpha-amylase, for example fromAlicyclobacillus pohliae, in particular Alicyclobacillus pohliaeNCIMB14276 strain. Variant polypeptides of the invention may be referredto as a “alpha-amylase variant”, an “improved alpha-amylase” and thelike.

The improved property will typically be a property with relevance to theuse of the variant alpha-amylase in the preparation of a baked product.

An alpha-amylase variant with an improved property relevant for a bakedproduct making may demonstrate reduced of hardness after storage of abaked product and/or reduced loss of resilience over storage of a bakedproduct.

The improved property may include increased strength of the dough,increased elasticity of the dough, increased stability of the dough,reduced stickiness of the dough, improved extensibility of the dough,improved machineability of the dough, increased volume of the bakedproduct, improved flavour of the baked product, improved crumb structureof the baked product, improved crumb softness of the baked product,reduced blistering of the baked product, improved crispiness, improvedresilience both initial and in particular after storage, reducedhardness after storage and/or improved anti-staling of the bakedproduct.

The improved property may include faster dough development time of thedough and/or reduced dough stickiness of the dough.

The improved property may include improved foldability of the bakedproduct, such as improved foldability of a tortilla, a pancake, a flatbread, a pizza crust, a roti and/or a slice of bread.

The improved property may include improved flexibility of the bakedproduct including improved flexibility of a tortilla, a pancake, a flatbread, a pizza crust, a roti and/or a slice of bread.

The improved property may include improved stackability of flat bakedproducts including tortillas, pancakes, flat breads, pizza crusts, roti.

The improved property may include reduced stickiness of noodles and/orincreased flexibility of noodles.

The improved property may include reduced clumping of cooked noodlesand/or improved flavor of noodles even after a period of storage.

The improved property may include reduction of formation of hairlinecracks in a product in crackers as well as creating a leavening effectand improved flavor development.

The improved property may include improved mouth feel and/or improvedsoftness on squeeze,

The improved property may include reduced damage during transport,including reduced breaking during transport.

The improved property may include reduced hardness after storage ofgluten-free bread.

The improved property may include improved resilience of gluten-freebread. The improved property may include improved resilience bothinitial and in particular after storage of gluten-free bread.

The improved property may include reduced hardness after storage of ryebread.

The improved property may include reduced loss of resilience overstorage of rye bread.

The improved property may include improved slice ability. This may bedemonstrated by observing the amount of crumbs after slicing. Lesscrumbs indicate a better slice ability

The improved property may include improved crumb structure and/orresilience, without creating gumminess.

The improved property may include reduced loss of resilience overstorage of a baked product comprising at least 5 wt % sugar, in anaspect comprising at least 8 wt % sugar, in an aspect comprising atleast 12 wt % sugar, in an aspect comprising at least 15 wt % sugarbased on flour. In an aspect comprising at least 18 wt % sugar, in anaspect comprising at least 20 wt % sugar, in an aspect comprising atleast 25 wt % sugar, in an aspect comprising at least 30 wt % sugarbased on flour. Herein 5 wt % sugar means 50 grams sugar per 1000 gramof flour used in the recipe, etc.

The improved property may include reduced hardness after storage of abaked product comprising at least 5 wt % sugar, in an aspect comprisingat least 8 wt % sugar, in an aspect comprising at least 12 wt % sugar,in an aspect comprising at least 15 wt % sugar based on flour. In anaspect comprising at least 18 wt % sugar, in an aspect comprising aspectat least 20 wt % sugar, in an aspect comprising at least 25 wt % sugar,in an aspect comprising at least 30 wt % sugar based on flour. Herein 5wt % sugar means 50 grams sugar per 1000 gram of flour used in therecipe, etc.

Each of these improvements may be determined as compared with areference polypeptide. The improved property may be demonstrated bypreparing a baked product comprising the alpha-amylase variant andanother comprising a parent polypeptide and comparing the results.

The improved property may be demonstrated in an assay or (bio)chemicalanalysis.

In particular, a variant alpha-amylase of the invention may showimproved productivity in comparison with a reference polypeptide.Alternatively, or in addition, a variant alpha-amylase of the inventionmay show an altered, such as reduced or increased, temperature stabilityor an altered activity at pH relevant for the baked product makingprocess, such as a lower pH or a higher pH, as compared with a referencepolypeptide.

The improved property may include:

-   -   increased (thermo)stability in comparison with a parent        polypeptide having alpha-amylase activity,    -   increased specific activity in comparison with a parent        polypeptide having alpha-amylase activity,    -   increased sucrose tolerance in comparison with a parent        polypeptide having alpha-amylase activity,    -   increased stability/activity at different pH range in comparison        with a parent polypeptide having alpha-amylase activity,    -   change in product spectrum (defined as ration of one product        over another) in comparison with a parent polypeptide having        alpha-amylase activity,    -   increased activity on raw starch in comparison with a parent        polypeptide having alpha-amylase activity,    -   altered temperature optimum,    -   alter substrate specificity, or    -   increased productivity in the production of the alpha-amylase        variant; in comparison with a parent polypeptide having        alpha-amylase activity.

In an aspect of the invention, there is provided a variant polypeptidehaving alpha-amylase activity, wherein the variant has an amino acidsequence which, when aligned with the alpha-amylase comprising thesequence set out in SEQ ID NO: 2, comprises at least one substitution ofan amino acid residue corresponding to any of amino acids

-   1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19,    20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,    37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 71,    72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,    89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,    105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,    118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,    131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,    144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,    157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,    170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,    183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,    196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,    209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,    222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,    235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247,    248, 249, 250, 251, 251, 253, 254, 255, 256, 257, 258, 259, 260,    261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273,    274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,    287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299,    300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312,    313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325,    326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,    339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351,    351, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364,    365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377,    378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390,    391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403,    404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416,    417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429,    430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442,    443, 444, 445, 446, 447, 448, 449, 450, 451, 451, 453, 454, 455,    456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468,    469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481,    482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494,    495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507,    508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520,    521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533,    534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546,    547, 548, 549, 550, 551, 551, 553, 554, 555, 556, 557, 558, 559,    560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572,    573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585,    586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598,    599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611,    612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624,    625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637,    638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650,    651, 651, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663,    664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676,    677, 678, 679, 680, 681, 682, 683, 684, 685, 686,

said positions being defined with reference to SEQ ID NO: 2 and whereinthe variant has one or more altered properties as compared with areference polypeptide having alpha-amylase activity.

In an embodiment according to the invention there is provided a variantpolypeptide having alpha-amylase activity, wherein the variant has anamino acid sequence which, when aligned with the alpha-amylasecomprising the sequence set out in SEQ ID NO: 2, comprises at least onesubstitution of an amino acid residue corresponding to any of aminoacids

4, 6, 13, 14, 15, 16, 20, 45, 47, 51, 54, 61, 68, 69, 70, 71, 72, 73,74, 75, 77, 78, 80, 82, 87, 88, 94, 95, 100, 103, 104, 117, 124, 125,126, 128, 130, 133, 134, 136, 143, 144, 146, 168, 174, 177, 178, 183,186, 188, 189, 190, 194, 195, 199, 200, 201, 204, 207, 210, 214, 217,219, 222, 225, 227, 233, 234, 235, 236, 240, 251, 252, 254, 258, 259,260, 261, 262, 263, 264, 266, 267, 269, 271, 273, 281, 282, 283, 284,286, 288, 299, 322, 323, 325, 327, 328, 331, 334, 350, 356, 358, 367,370, 371, 374, 377, 378, 388, 391, 414, 421, 422, 445, 450, 467, 488,505, 536, 548, 583, 588, 603, 637, 648, 651, 652, 660, 676, 677,

said positions being defined with reference to SEQ ID NO: 2 and whereinthe variant has one or more altered properties as compared with areference polypeptide having alpha-amylase activity.

The invention also provides:

-   -   a nucleic acid sequence encoding a variant of the invention;    -   a nucleic acid construct comprising such a nucleic acid sequence        operably linked to one or more control sequences capable of        directing the expression of an alpha-amylase in a suitable        expression host;    -   a recombinant expression vector comprising such a nucleic acid        construct; and    -   a recombinant host cell comprising such an expression vector.

The invention also relate to a method for producing an alpha-amylasecomprising cultivating the host cell of the invention under conditionsconducive to production of the alpha-amylase and recovering thealpha-amylase.

Also, the invention relates to a method of producing an alpha-amylasepolypeptide variant, which method comprises:

a) selecting a polypeptide having alpha-amylase activity;

b) substituting at least one amino acid residue corresponding to any ofamino acids

4, 6, 13, 14, 15, 16, 20, 45, 47, 51, 54, 61, 68, 69, 70, 71, 72, 73,74, 75, 77, 78, 80, 82, 87, 88, 94, 95, 100, 103, 104, 117, 124, 125,126, 128, 130, 133, 134, 136, 143, 144, 146, 168, 174, 177, 178, 183,186, 188, 189, 190, 194, 195, 199, 200, 201, 204, 207, 210, 214, 217,219, 222, 225, 227, 233, 234, 235, 236, 240, 251, 252, 254, 258, 259,260, 261, 262, 263, 264, 266, 267, 269, 271, 273, 281, 282, 283, 284,286, 288, 299, 322, 323, 325, 327, 328, 331, 334, 350, 356, 358, 367,370, 371, 374, 377, 378, 388, 391, 414, 421, 422, 445, 450, 467, 488,505, 536, 548, 583, 588, 603, 637, 648, 651, 652, 660, 676, 677,

said positions being defined with reference to SEQ ID NO: 2;

c) optionally substituting one or more further amino acids as defined inb);

d) preparing the variant resulting from steps a)-c);

e) determining a property of the variant; and

f) selecting a variant having an altered property in comparison to thealpha-amylase comprising the sequence set out in SEQ ID NO: 2, therebyto produce an alpha-amylase polypeptide variant.

Further the invention relates to:

-   -   a composition comprising the variant of the invention or        obtainable by a method of the invention;    -   use of a variant alpha-amylase according to the invention or of        a composition of the invention in the preparation of a baked        product;    -   a process for the production of a baked product, which method        comprises comprising adding an effective amount of a variant        polypeptide according to the invention of a composition        according to the invention to dough and carrying out appropriate        further baking manufacturing steps; and    -   a baked product obtainable by such process or use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Sets out the plasmid map op pGBB09, the plasmid is used toconstruct the expression vectors for alpha-amylase variants.

FIG. 2. Sets out the plasmid map of pGBB09DSM-AM1 containing the DSM-AMgene that is used for the production of a reference alpha-amylase.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 sets out the polynucleotide sequence from Alicyclobacilluspohliae NCIMB14276 encoding the wild type signal sequence (set out innucleotides 1 to 99), the wild-type alpha-amylase polypeptide (set outin nucleotides 100 to 2157), and a stop codon at the 3′-terminus (setout in nucleotides 2157 to 2160).

SEQ ID NO: 2 sets out the amino acid sequence of the Alicyclobacilluspohliae NCIMB14276 wild type alpha-amylase polypeptide.

SEQ ID NO: 3 sets out a synthetic DNA fragment containing the PmeIrestriction site, the amyQ terminator and the SphI and HindIIIrestriction site.

SEQ ID NO: 4 sets out a synthetic DNA fragment containing a ribosomebinding site and PacI restriction site.

SEQ ID NO: 5 sets out a synthetic DNA fragment containing a double stopcodon and PmeI restriction site.

SEQ ID NO 6: sets out the polynucleotide sequence of a synthetic DNAconstruct exciting of a PacI site, ribosome binding site, wild typeDSM-AM sequence as set out in SEQ ID NO:1, double stop codon and PmeIrestriction site.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

Throughout the present specification and the accompanying claims, thewords “comprise”, “include” and “having” and variations such as“comprises”, “comprising”, “includes” and “including” are to beinterpreted inclusively. That is, these words are intended to convey thepossible inclusion of other elements or integers not specificallyrecited, where the context allows.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to one or at least one) of the grammatical object of thearticle. By way of example, “an element” may mean one element or morethan one element.

Alpha-Amylase Activity

The alpha-amylase variant according to the invention and the parentpolypeptide herein are a starch degrading enzymes. The alpha-amylasevariant according to the invention and the parent polypeptide hereinhave alpha-amylase activity. Alpha-amylase activity can suitably bedetermined using the Ceralpha® procedure, which is recommended by theAmerican Association of Cereal Chemists (AACC).

NBAU Activity

Enzymatic activity of a alpha-amylase variant and of a parentpolypeptide may be expressed as NBAU. NBAU activity can suitably bedetermined using the NBAU assay as described herein.

Pre-Mix

The term “pre-mix” is defined herein to be understood in itsconventional meaning, i.e. as a mix of baking agents, generallyincluding flour, which may be used not only in industrial bread-bakingplants/facilities, but also in retail bakeries. The pre-mix may beprepared by mixing the alpha-amylase polypeptide and the G4-formingamylase or the enzyme composition according to the invention with asuitable carrier such as flour, starch or a salt. The pre-mix maycontain additives as mentioned herein.

Baked Product

The term ‘baked product’ refers to a baked food product prepared from adough.

Examples of baked products, whether of a white, brown or whole-mealtype, which may be advantageously produced by the present inventioninclude bread (in particular white, whole-meal or rye bread), typicallyin the form of loaves or rolls, French baguette-type bread, pastries,croissants, brioche, panettone, pasta, noodles (boiled or (stir-)fried),pita bread and other flat breads, tortillas, tacos, cakes, pancakes,cookies in particular biscuits, doughnuts, including yeasted doughnuts,bagels, pie crusts, steamed bread, crisp bread, brownies, sheet cakes,snack foods (e.g., pretzels, tortilla chips, fabricated snacks,fabricated potato crisps). The term baked product includes, breadcontaining from 2 to 30 wt % sugar, fruit containing bread, breakfastcereals, cereal bars, eggless cake, soft rolls and gluten-free bread.Gluten free bread herein and herein after is bread than contains at most20 ppm gluten. Several grains and starch sources are consideredacceptable for a gluten-free diet. Frequently used sources are potatoes,rice and tapioca (derived from cassava) Baked product includes withoutlimitation tin bread, loaves of bread, twists, buns, such as hamburgerbuns or steamed buns, chapati, rusk, dried steam bun slice, bread crumb,matzos, focaccia, melba toast, zwieback, croutons, soft pretzels, softand hard bread, bread sticks, yeast leavened and chemically-leavenedbread, laminated dough products such as Danish pastry, croissants orpuff pastry products, muffins, danish, bagels, confectionery coatings,crackers, wafers, pizza crusts, tortillas, pasta products, crepes,waffles, parbaked products and refrigerated and frozen dough products.

An example of a parbaked product includes, without limitation, partiallybaked bread that is completed at point of sale or consumption with ashort second baking process.

The bread may be white or brown pan bread; such bread may for example bemanufactured using a so called American style Sponge and Dough method oran American style Direct method.

The term tortilla herein includes corn tortilla and wheat tortilla. Acorn tortilla is a type of thin, flat bread, usually unleavened madefrom finely ground maize (usually called “corn” in the United States). Aflour tortilla is a type of thin, flat bread, usually unleavened, madefrom finely ground wheat flour. The term tortilla further includes asimilar bread from South America called arepa, though arepas aretypically much thicker than tortillas. The term tortilla furtherincludes a laobing, a pizza-shaped thick “pancake” from China and anIndian Roti, which is made essentially from wheat flour. A tortillausually has a round or oval shape and may vary in diameter from about 6to over 30 cm.

Dough

The term “dough” is defined herein as a mixture of flour and otheringredients. In one aspect the dough is firm enough to knead or roll.The dough may be fresh, frozen, prepared or parbaked. The preparation offrozen dough is described by Kulp and Lorenz in Frozen and RefrigeratedDoughs and Batters.

Dough is made using dough ingredients, which include without limitation(cereal) flour, a lecithin source including egg, water, salt, sugar,flavours, a fat source including butter, margarine, oil and shortening,baker's yeast, chemical leavening systems such as a combination of anacid (generating compound) and bicarbonate, a protein source includingmilk, soy flour, oxidants (including ascorbic acid, bromate andAzodicarbonamide (ADA)), reducing agents (including L-cysteine),emulsifiers (including mono/di glycerides, monoglycerides such asglycerol monostearate (GMS), sodium stearoyl lactylate (SSL), calciumstearoyl lactylate (CSL), polyglycerol esters of fatty acids (PGE) anddiacetyl tartaric acid esters of mono- and diglycerides (DATEM), gums(including guargum and xanthangum), flavours, acids (including citricacid, propionic acid), starch, modified starch, gluten, humectants(including glycerol) and preservatives.

Cereals include maize, rice, wheat, barley, sorghum, millet, oats, rye,triticale, buckwheat, quinoa, spelt, einkorn, emmer, durum and kamut.

Dough is usually made from basic dough ingredients including (cereal)flour, such as wheat flour or rice flour, water and optionally salt. Forleavened products, primarily baker's yeast is used next to chemicalleavening systems such as a combination of an acid (generating compound)and bicarbonate.

The term dough herein includes a batter. A batter is a semi-liquidmixture, being thin enough to drop or pour from a spoon, of one or moreflours combined with liquids such as water, milk or eggs used to preparevarious foods, including cake.

The dough may be made using a mix including a cake mix, a biscuit mix, abrownie mix, a bread mix, a pancake mix and a crepe mix.

The term dough includes frozen dough, which may also be referred to asrefrigerated dough. There are different types of frozen dough; thatwhich is frozen before proofing and that which is frozen after a partialor complete proofing stage. The frozen dough is typically used formanufacturing baked products including without limitation biscuits,breads, bread sticks and croissants.

A gene or cDNA coding for an alpha-amlyase or pro-alpha-amylase, forexample a variant of the invention, may be cloned and over-expressed ina host organism. Well known host organisms that have been used foralphaamylase over-expression in the past include Aspergillus, Kluyveromyces,Trichoderma, Escherichia coli, Pichia, Saccharomyces, Yarrowia,Neurospora or Bacillus.

The alpha-amylase variant may be manufactured industrially usingrecombinant DNA technology, e.g. using filamentous fungi such asAspergillus species, yeast strains, e.g. of Klyuveromyces species, orbacterial species, e.g. E. coli, as host organisms. Such recombinantmicrobial production strains are constructed and continuously improvedusing DNA technology as well as classical strain improvement measuresdirected towards optimising the expression and secretion of aheterologous protein.

In the invention, an alpha-amylase variant may be provided in the formof prealpha-amylase variant or (mature) alpha-amylase variant. Acorresponding nucleic acid sequence may also be provided, i.e. apolynucleotide that encodes a pre-alpha-amylase or a (mature)alpha-amylase may be provided.

Herein, positions which may be substituted to achieve a variant of theinvention are defined with reference to SEQ ID NO: 2 which is a maturealpha-amylase, i.e. it is a sequence which does not include apresequence.

The invention concerns variant polypeptides having alpha-amylaseactivity as compared with a reference polypeptide having alpha-amylaseactivity. The reference polypeptide may typically be a wild-typepolypeptide having alpha-amylase activity, such as the alpha-amylase ofSEQ ID NO: 2. The reference polypeptide may also be referred to as aparent polypeptide or comparison polypeptide.

More concretely, the invention relates to a variant polypeptide havingalpha-amylase activity, wherein the variant has an amino acid sequencewhich, when aligned with the alpha-amylase comprising the sequence setout in SEQ ID NO: 2, comprises at least one substitution of an aminoacid residue corresponding to any of amino acids

-   1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19,    20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,    37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 71,    72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,    89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,    105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,    118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,    131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,    144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,    157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,    170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,    183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,    196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,    209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,    222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,    235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247,    248, 249, 250, 251, 251, 253, 254, 255, 256, 257, 258, 259, 260,    261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273,    274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,    287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299,    300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312,    313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325,    326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,    339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351,    351, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364,    365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377,    378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390,    391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403,    404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416,    417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429,    430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442,    443, 444, 445, 446, 447, 448, 449, 450, 451, 451, 453, 454, 455,    456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468,    469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481,    482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494,    495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507,    508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520,    521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533,    534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546,    547, 548, 549, 550, 551, 551, 553, 554, 555, 556, 557, 558, 559,    560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572,    573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585,    586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598,    599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611,    612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624,    625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637,    638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650,    651, 651, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663,    664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676,    677, 678, 679, 680, 681, 682, 683, 684, 685, 686,

said positions being defined with reference to SEQ ID NO: 2 and whereinthe variant has one or more altered properties as compared with areference polypeptide having alpha-amylase activity.

A wild type reference polypeptide may be obtained from any suitableorganisms.

Suitable wild type reference polypeptides may be obtained formAlicyclobacillus pohliae NCIMB14276.

Preferably, the reference polypeptide is the alpha amylase set out inSEQ ID NO: 2.

The parent polypeptide having alpha-amylase activity is preferably isthe alpha amylase set out in SEQ ID NO: 2.

A variant polypeptide will typically have an improved property ascompared to a reference polypeptide, in particular with respect to aproperty relevant to the use of the variant polypeptide in baked productmaking.

Improved productivity may be demonstrated by an alpha-amylase variantthat shows improved expression as compared with a parent polypeptide.

The improved property will typically be a property with relevance to theuse of the variant alpha-amylase in the preparation of a baked product.

An alpha-amylase variant with an improved property relevant for a bakedproduct making may demonstrate reduced of hardness after storage of abaked product and/or reduced loss of resilience over storage of a bakedproduct.

The improved property may include increased strength of the dough,increased elasticity of the dough, increased stability of the dough,reduced stickiness of the dough, improved extensibility of the dough,improved machineability of the dough, increased volume of the bakedproduct, improved flavour of the baked product, improved crumb structureof the baked product, improved crumb softness of the baked product,reduced blistering of the baked product, improved crispiness, improvedresilience both initial and in particular after storage, reducedhardness after storage and/or improved anti-staling of the bakedproduct.

The improved property may include faster dough development time of thedough and/or reduced dough stickiness of the dough.

The improved property may include improved foldability of the bakedproduct, such as improved foldability of a tortilla, a pancake, a flatbread, a pizza crust, a roti and/or a slice of bread.

The improved property may include improved flexibility of the bakedproduct including improved flexibility of a tortilla, a pancake, a flatbread, a pizza crust, a roti and/or a slice of bread.

The improved property may include improved stackability of flat bakedproducts including tortillas, pancakes, flat breads, pizza crusts, roti.

The improved property may include reduced stickiness of noodles and/orincreased flexibility of noodles.

The improved property may include reduced clumping of cooked noodlesand/or improved flavor of noodles even after a period of storage.

The improved property may include reduction of formation of hairlinecracks in a product in crackers as well as creating a leavening effectand improved flavor development.

The improved property may include improved mouth feel and/or improvedsoftness on squeeze,

The improved property may include reduced damage during transport,including reduced breaking during transport.

The improved property may include reduced hardness after storage ofgluten-free bread.

The improved property may include improved resilience of gluten-freebread. The improved property may include improved resilience bothinitial and in particular after storage of gluten-free bread.

The improved property may include reduced hardness after storage of ryebread.

The improved property may include reduced loss of resilience overstorage of rye bread,

The improved property may include reduced loss of resilience overstorage of a baked product comprising at least 5 wt % sugar, in anaspect comprising at least 8 wt % sugar, in an aspect comprising atleast 12 wt % sugar, in an aspect comprising at least 15 wt % sugarbased on flour. In an aspect comprising at least 18 wt % sugar, in anaspect comprising at least 20 wt % sugar, in an aspect comprising atleast 25 wt % sugar, in an aspect comprising at least 30 wt % sugarbased on flour. Herein 5 wt % sugar means 50 grams sugar per 1000 gramsof flour used in the recipe.

The improved property may include reduced hardness after storage of abaked product comprising at least 5 wt % sugar, in an aspect comprisingat least 8 wt % sugar, in an aspect comprising at least 12 wt % sugar,in an aspect comprising at least 15 wt % sugar based on flour. In anaspect comprising at least 18 wt % sugar, in an aspect comprising aspectat least 20 wt % sugar, in an aspect comprising at least 25 wt % sugar,in an aspect comprising at least 30 wt % sugar based on flour. Herein 5wt % sugar means 50 grams sugar per 1000 grams of flour used in therecipe, etc.

Each of these improvements may be determined as compared with areference polypeptide. The improved property may be demonstrated bypreparing a baked product comprising the alpha-amylase variant andanother comprising a parent polypeptide and comparing the results.

The improved property may be demonstrated in an assay or (bio)chemicalanalysis.

In particular, a variant alpha-amylase of the invention may showimproved productivity in comparison with a reference polypeptide.Alternatively, or in addition, a variant alpha-amylase of the inventionmay show an altered, such as reduced or increased, temperature stabilityor an altered activity at pH relevant for the baked product makingprocess, such as a lower pH or a higher pH, as compared with a referencepolypeptide.

The improved property may include:

-   -   increased (thermo)stability in comparison with a parent        polypeptide having alpha-amylase activity,    -   increased specific activity in comparison with a parent        polypeptide having alpha-amylase activity,    -   increased sucrose tolerance in comparison with a parent        polypeptide having alpha-amylase activity,    -   increased stability/activity at different pH range in comparison        with a parent polypeptide having alpha-amylase activity,    -   change in product spectrum (defined as ration of one product        over another) in comparison with a parent polypeptide having        alpha-amylase activity,    -   increased activity on raw starch in comparison with a parent        polypeptide having alpha-amylase activity,    -   altered temperature optimum,    -   alter substrate specificity, or    -   increased productivity in the production of the alpha-amylase        variant; in comparison with a parent polypeptide having        alpha-amylase activity.

Thermostability may be determined by measuring the residual activityafter incubation at a higher temperature (e.g. 50-100° C. for 1-20 min),using a suitable activity assay (such as Ceralpha) or alternatively theNBAU assay as described herein.

Specific activity may be determined by measuring the activity per mg ofprotein (amount of protein can e.g. be estimated from SDS-PAGE, or ifsample is pure enough can be determined using Bradford assay).

Sucrose tolerance may be determined by measuring the activity in thepresence of increasing concentration of sucrose (for example incubatewith Phadebas tablets for 15 min at 60° C. in the presence of 0-40% (byweight) sucrose. Express as a percentage of the activity at 0% sucrose.)The activity may be determined using a suitable activity assay (such asCeralpha) or alternatively the NBAU assay as described herein.

pH stability may be determined by measuring the thermostability in a pHrange (measure thermostability as described above, but do incubation ina range of pH values, e.g. 2-12)

Activity at different pH (determine pH optimum) may be determined bymeasuring the activity in a pH range (e.g. 2-12)

Product spectrum may be determined by measuring the amount of differentoligosaccharides formed from starch, for example using HPLC.

Activity on raw starch may be determined by incubating the enzyme with asuspension of native starch (e.g. wheat or maize), followed bycentrifuging to remove starch granules, and determining the amount ofreducing sugars released (e.g. with DNS method)

Altered temperature optimum may be determined by measuring activity asdescribed above over a temperature range (e.g. 50-100° C.).

Substrate specificity may be determined by measuring activity asdescribed above on different substrates.

The present invention also relates to methods for preparing a dough or abaked product comprising incorporating into the dough an effectiveamount of the alpha-amylase variant, which improves one or moreproperties of the dough or the baked product obtained from the doughrelative to a dough or a baked product in which a parent polypeptide isincorporated.

The phrase “incorporating into the dough” is defined herein as addingthe alpha-amylase variant or a parent polypeptide to the dough, anyingredient from which the dough is to be made, and/or any mixture ofdough ingredients from which the dough is to be made. In other words,the alpha-amylase variant or a parent polypeptide to the dough may beadded in any step of the dough preparation and may be added in one, twoor more steps. The alpha-amylase variant or a parent polypeptide to thedough are added to the ingredients of a dough that is kneaded and bakedto make the baked product using methods well known in the art. See, forexample, U.S. Pat. No. 4,567,046, EP-A-426,211, JP-A-60-78529,JP-A-62-111629, and JP-A-63-258528.

The term “effective amount” is defined herein as an amount of thealpha-amylase variant that is sufficient for providing a measurableeffect on at least one property of interest of the dough and/or bakedproduct. A suitable amount of alpha-amylase variant is in a range of0.5-1500 NBAU/kg flour, in an embodiment 5-200 NBAU/kg flour, in afurther embodiment 20-100 NBAU/kg flour. A suitable amount includes 1ppm-2000 ppm of an enzyme having an activity in a range of about 700 to1100 NBAU/g. In an embodiment an effective amount is in a range of10-200 ppm of an enzyme having an activity in a range of about 700 to1100 NBAU/g, in another embodiment 30-100 ppm of an enzyme having anactivity in a range of about 700 to 1100 NBAU/g. In an embodiment aneffective amount is in a range of 10-200 ppm of an enzyme having anactivity of about 700 to 1100 NBAU/g. Herein and hereinafter NBAU standsfor New Baking Amylase Unit as defined in the examples under the headingNBAU Assay

The term “improved property” is defined herein as any property of adough and/or a product obtained from the dough, particularly a bakedproduct, which is improved by the action of the alpha-amylase variant,the composition according to the invention or the pre-mix according tothe invention relative to a dough or product in which a parentpolypeptide is incorporated. The improved property may include, but isnot limited to, increased strength of the dough, increased elasticity ofthe dough, increased stability of the dough, reduced stickiness of thedough, improved extensibility of the dough, improved machineability ofthe dough, increased volume of the baked product, improved flavour ofthe baked product, improved crumb structure of the baked product,improved crumb softness of the baked product, reduced blistering of thebaked product, improved crispiness, improved resilience both initial andin particular after storage, reduced hardness after storage and/orimproved anti-staling of the baked product.

The improved property may include faster dough development time of thedough and/or reduced dough stickiness of the dough.

The improved property may include improved foldability of the bakedproduct, such as improved foldability of a tortilla, a pancake, a flatbread, a pizza crust, a roti and/or a slice of bread.

The improved property may include improved flexibility of the bakedproduct including improved flexibility of a tortilla, a pancake, a flatbread, a pizza crust, a roti and/or a slice of bread.

The improved property may include improved stackability of flat bakedproducts including tortillas, pancakes, flat breads, pizza crusts, roti.

The improved property may include reduced stickiness of noodles and/orincreased flexibility of noodles.

The improved property may include reduced clumping of cooked noodlesand/or improved flavor of noodles even after a period of storage.

The improved property may include reduction of formation of hairlinecracks in a product in crackers as well as creating a leavening effectand improved flavor development.

The improved property may include improved mouth feel and/or improvedsoftness on squeeze,

The improved property may include reduced damage during transport,including reduced breaking during transport.

The improved property may include reduced hardness after storage ofgluten-free bread.

The improved property may include improved resilience of gluten-freebread. The improved property may include improved resilience bothinitial and in particular after storage of gluten-free bread.

The improved property may include reduced hardness after storage of ryebread.

The improved property may include reduced loss of resilience overstorage of rye bread,

The improved property may include reduced loss of resilience overstorage of a baked product comprising at least 5 wt % sugar, in anaspect comprising at least 8 wt % sugar, in an aspect comprising atleast 12 wt % sugar, in an aspect comprising at least 15 wt % sugarbased on flour. In an aspect comprising at least 18 wt % sugar, in anaspect comprising at least 20 wt % sugar, in an aspect comprising atleast 25 wt % sugar, in an aspect comprising at least 30 wt % sugarbased on flour. Herein 5 wt % sugar means 50 grams sugar per 1000 gramof flour used in the recipe, etc.

The improved property may include reduced hardness after storage of abaked product comprising at least 5 wt % sugar, in an aspect comprisingat least 8 wt % sugar, in an aspect comprising at least 12 wt % sugar,in an aspect comprising at least 15 wt % sugar based on flour. In anaspect comprising at least 18 wt % sugar, in an aspect comprising aspectat least 20 wt % sugar, in an aspect comprising at least 25 wt % sugar,in an aspect comprising at least 30 wt % sugar based on flour. Herein 5wt % sugar means 50 grams sugar per 1000 gram of flour used in therecipe, etc.

Improved mouth feel includes sense of softness on an initial bite orafter chewing, preferably without a sticky feeling in the mouth and/orwithout the baked product sticking to the teeth. Improved mouth feelincludes the baked product feeling less dry in the mouth on an initialbite or after chewing. Improved mouth feel includes the baked productfeeling less dry in the mouth on an initial bite or after chewing afterit has been kept outside its packaging or container. The improvedproperty may include that after a slice of bread was taken from itspackaging or container and exposed to ambient conditions for 5 minutes,in an aspect for 10 minutes, in an aspect for 20 minutes it has improvedmouthfeel.

The improved property may include that after a the cookie was taken fromits packaging or container and exposed to ambient conditions for 10minutes, in an aspect for 20 minutes, in an aspect for 30 minutes, in anaspect an hour it has improved mouthfeel.

In an aspect ambient conditions herein and herein after include atemperature of 20 degrees C. and a moisture level of 40% humidity.

Reduced breaking during transport includes the baked product, includingwithout limitation cookies, bread such as gluten free bread, does notbreak in additional pieces as a consequence of transport.

Improved softness on squeeze includes the tactile experience that if abun is held between the fingers and the thumb of a hand and the thumband fingers are moved towards each other it takes less force.

Improved foldability of a baked product may be determined as follows.

The baked product is laid on a flat surface. The baked product is foldedby picking up one edge of the product and placing it on the oppositeedge of the product. This way a folded baked product is obtained havinga bend curve in an area located at or close to the center. The surfaceof the outside of the bend of folded baked product is visuallyinspected. The foldability is improved if fewer cracks are observed ator close to the bend. This may be a particularly useful property if thebaked product is a tortilla and/or a slice of bread.

Improved stackability may be determined as follows.

10 baked products are stacked on top of each other and sealed in apolymer package, such as polyethylene foil. This yields a pack of bakedproducts. 10 packs of baked product are stacked on top of each other andkept under ambient conditions for 3 days, in an aspect for 5 days in anaspect for 1 week, in an aspect for 2 weeks. Ambient conditions areconditions as defined herein. After this period the bottom pack of bakedproducts is opened, the baked products are separated from each other andthe surfaces of the products are visually inspected. The stackability isimproved if less surface damage is observed. Surface damage may becaused e.g. by rupture of the surface during separation of two bakedproducts that were stacked on top of each other. This may be aparticularly useful property if the baked product is a tortilla.

Faster dough development time may be determined as follows

Dough development time is the time the dough need to reach maximumconsistency, maximum viscosity before gluten strands begin to breakdown. It may be determined by measuring peak time, using a Farinograph®from Brabender®, Germany. If a faronigraph is used to determine doughdevelopment time, dough development time is the time between the momentwater is added and the moment the curve reaches its highest point. Peaktime is preferably expressed in minutes.

Reduced dough stickiness may be determined as follows.

Dough stickiness is preferably determined on two separate batches of atleast 8 dough pieces, with the Texture Analyser TAXT2i (Stable MicroSystems Ltd., Surrey, UK) equipped with a 5 kg load cell in the measureforce in compression mode with a cylindrical probe (25 mm diameter).Using pre- and post-test speeds of 2.0 mm/s, while the test speed is 1.0mm/s. Dough pieces are centered and compressed 50% and the probe is heldfor 10 s at maximum compression. A negative peak value indicates doughstickiness. A less negative peak value indicates reduced doughstickiness.

Increased flexibility may be determined as follows.

The baked product is laid on a flat surface. The baked product is rolledto a shape similar to a pipe, this way a rolled baked product isobtained. The flexiblity is improved if the rolled baked product remainsits rolled up shape and does not roll open. This may be a particularlyuseful property if the baked product is a tortilla or a pancake.

The improved property may be determined by comparison of a dough and/ora baked product prepared with and without addition of the (isolated)polypeptide of the present invention in accordance with the methods ofpresent invention which are described below in the Examples.Organoleptic qualities may be evaluated using procedures wellestablished in the baking industry, and may include, for example, theuse of a panel of trained taste-testers.

The term “increased strength of the dough” is defined herein as theproperty of a dough that has generally more elastic properties and/orrequires more work input to mould and shape.

The term “increased elasticity of the dough” is defined herein as theproperty of a dough which has a higher tendency to regain its originalshape after being subjected to a certain physical strain.

The term “increased stability of the dough” is defined herein as theproperty of a dough that is less susceptible to forming faults as aconsequence of mechanical abuse thus better maintaining its shape andvolume and is evaluated by the ratio of height:width of a cross sectionof a loaf after normal and/or extended proof.

The term “reduced stickiness of the dough” is defined herein as theproperty of a dough that has less tendency to adhere to surfaces, e.g.,in the dough production machinery, and is either evaluated empiricallyby the skilled test baker or measured by the use of a texture analyser(e.g. a TAXT Plus) as known in the art.

The term “improved extensibility of the dough” is defined herein as theproperty of a dough that can be subjected to increased strain orstretching without rupture.

The term “improved machineability of the dough” is defined herein as theproperty of a dough that is generally less sticky and/or more firmand/or more elastic. Consequently there is less fouling of plantequipment and a reduced need for cleaning.

The term “increased volume of the baked product” is preferably measuredas the volume of a given loaf of bread determined by an automated breadvolume analyser (eg. BVM-3, TexVol Instruments AB, Viken, Sweden), usingultrasound or laser detection as known in the art. In case the volume isincreased, the property is improved. Alternatively the height of thebaked product after baking in the same size tin is an indication of thebaked product volume. In case the height of the baked product hasincreased, the volume of the baked product has increased.

The term “reduced blistering of the baked product” is defined herein asa visually determined reduction of blistering on the crust of the bakedbread.

The term “improved crumb structure of the baked product” is definedherein as the property of a baked product with finer cells and/orthinner cell walls in the crumb and/or more uniform/homogenousdistribution of cells in the crumb and is usually evaluated visually bythe baker or by digital image analysis as known in the art (eg. C-cell,Calibre Control International Ltd, Appleton, Warrington, UK).

The term “improved softness of the baked product” is the opposite of“hardness” and is defined herein as the property of a baked product thatis more easily compressed and is evaluated either empirically by theskilled test baker or measured by the use of a texture analyzer (e.g.TAXT Plus) as known in the art.

The term “improved flavor of the baked product” is evaluated by atrained test panel.

The term “improved anti-staling of the baked product” is defined hereinas the properties of a baked product that have a reduced rate ofdeterioration of quality parameters, e.g. reduced hardness after storageand/or decreased loss of resilience after storage.

Anti-staling properties may be demonstrated by a reduced hardness afterstorage of the baked product. The enzyme composition according to theinvention or the pre-mix according to the invention may result inreduced hardness, e.g. in a baked product that is more easilycompressed. The hardness of the baked product may be evaluated eitherempirically by the skilled test baker or measured by the use of atexture analyzer (e.g. TAXT Plus) as known in the art. The hardnessmeasured within 24 hours after baking is called initial hardness. Thehardness measured 24 hours or more after baking is called hardness afterstorage, and is also a measure for determining shelf life. In case theinitial hardness has reduced, it has improved. In case the hardnessafter storage has reduced, it has improved. Preferably hardness ismeasured as described in example 9 herein. Resilience of the bakedproduct is preferably measured by the use of a texture analyzer (e.g.TAXTPlus) as known in the art.The resilience measured within 24 hours after baking is called initialresilience. The resilience measured 24 hours or more after baking iscalled resilience after storage, and is also a measure for determiningshelf life. Freshly baked product typically gives crumb of high initialresilience but resilience is lost over shelf-life. Improved anti-stalingproperties may be demonstrated by a reduced loss of resilience overstorage. Preferably resilience is measured as described in example 9herein.

The term “improved crispiness” is defined herein as the property of abaked product to give a crispier sensation than a reference product asknown in the art, as well as to maintain this crispier perception for alonger time than a reference product. This property can be quantified bymeasuring a force versus distance curve at a fixed speed in acompression experiment using e.g. a texture analyzer TA-XT Plus (StableMicro Systems Ltd, Surrey, UK), and obtaining physical parameters fromthis compression curve, viz. (i) force of the first peak, (ii) distanceof the first peak, (iii) the initial slope, (iv) the force of thehighest peak, (v) the area under the graph and (vi) the amount offracture events (force drops larger than a certain preset value).Indications of improved crispness are a higher force of the first peak,a shorter distance of the first peak, a higher initial slope, a higherforce of the highest peak, higher area under the graph and a largernumber of fracture events. A crispier product should score statisticallysignificantly better on at least two of these parameters as compared toa reference product. In the art, “crispiness” is also referred to ascrispness, crunchiness or crustiness, meaning a material with a crispy,crunchy or crusty fracture behaviour.

The present invention may provide a dough having at least one of theimproved properties selected from the group consisting of increasedstrength, increased elasticity, increased stability, reduced stickiness,and/or improved extensibility of the dough.

The invention also may provide a baked product having increased loafvolume. The invention may provide as well a baked product having atleast one improved property selected from the group consisting ofincreased volume, improved flavour, improved crumb structure, improvedcrumb softness, improved crispiness, reduced blistering and/or improvedanti-staling.

The enzyme composition according to the invention or the pre-mixaccording to the invention may be used for retarding staling of a bakedproduct such as bread and/or cake. Retarding of staling may be indicatedby a reduced hardness, in particular a reduced hardness after storagecompared to a baked product, including bread and cake, that is producedwith the alpha-amylase variant in comparison with a parent polypeptide.

In an aspect according to the invention, there is provided a variantpolypeptide having alpha-amylase activity, wherein the variant has anamino acid sequence which, when aligned with the alpha-amylasecomprising the sequence set out in SEQ ID NO: 2, comprises at least onesubstitution of an amino acid residue corresponding to any of aminoacids

-   1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19,    20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,    37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 71,    72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,    89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,    105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,    118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,    131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,    144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,    157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,    170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,    183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,    196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,    209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,    222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,    235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247,    248, 249, 250, 251, 251, 253, 254, 255, 256, 257, 258, 259, 260,    261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273,    274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,    287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299,    300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312,    313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325,    326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,    339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351,    351, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364,    365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377,    378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390,    391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403,    404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416,    417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429,    430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442,    443, 444, 445, 446, 447, 448, 449, 450, 451, 451, 453, 454, 455,    456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468,    469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481,    482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494,    495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507,    508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520,    521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533,    534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546,    547, 548, 549, 550, 551, 551, 553, 554, 555, 556, 557, 558, 559,    560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572,    573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585,    586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598,    599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611,    612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624,    625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637,    638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650,    651, 651, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663,    664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676,    677, 678, 679, 680, 681, 682, 683, 684, 685, 686,

said positions being defined with reference to SEQ ID NO: 2 and whereinthe variant has one or more altered properties as compared with areference polypeptide having alpha-amylase activity.

In an embodiment according to the invention there is provided a variantpolypeptide having alpha-amylase activity, wherein the variant has anamino acid sequence which, when aligned with the alpha-amylasecomprising the sequence set out in SEQ ID NO: 2, comprises at least onesubstitution of an amino acid residue corresponding to any of aminoacids

4, 6, 13, 14, 15, 16, 20, 45, 47, 51, 54, 61, 68, 69, 70, 71, 72, 73,74, 75, 77, 78, 80, 82, 87, 88, 94, 95, 100, 103, 104, 117, 124, 125,126, 128, 130, 133, 134, 136, 143, 144, 146, 168, 174, 177, 178, 183,186, 188, 189, 190, 194, 195, 199, 200, 201, 204, 207, 210, 214, 217,219, 222, 225, 227, 233, 234, 235, 236, 240, 251, 252, 254, 258, 259,260, 261, 262, 263, 264, 266, 267, 269, 271, 273, 281, 282, 283, 284,286, 288, 299, 322, 323, 325, 327, 328, 331, 334, 350, 356, 358, 367,370, 371, 374, 377, 378, 388, 391, 414, 421, 422, 445, 450, 467, 488,505, 536, 548, 583, 588, 603, 637, 648, 651, 652, 660, 676, 677,

said positions being defined with reference to SEQ ID NO: 2 and whereinthe variant has one or more altered properties as compared with areference polypeptide having alpha-amylase activity.

Table 1 sets out positions that may influence specific properties of thevariant alpha-amylases of the invention.

Accordingly, in an embodiment of the alpha-amylase variant according tothe invention comprises an amino acid sequence which, when aligned withthe sequence set out in SEQ ID NO: 2, comprises at least onesubstitution of an amino acid residue corresponding to any of aminoacids

-   1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19,    20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,    37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 71,    72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,    89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,    105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,    118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,    131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,    144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,    157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,    170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,    183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,    196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,    209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,    222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,    235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247,    248, 249, 250, 251, 251, 253, 254, 255, 256, 257, 258, 259, 260,    261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273,    274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,    287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299,    300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312,    313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325,    326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,    339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351,    351, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364,    365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377,    378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390,    391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403,    404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416,    417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429,    430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442,    443, 444, 445, 446, 447, 448, 449, 450, 451, 451, 453, 454, 455,    456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468,    469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481,    482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494,    495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507,    508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520,    521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533,    534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546,    547, 548, 549, 550, 551, 551, 553, 554, 555, 556, 557, 558, 559,    560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572,    573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585,    586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598,    599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611,    612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624,    625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637,    638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650,    651, 651, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663,    664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676,    677, 678, 679, 680, 681, 682, 683, 684, 685, 686,

said positions being defined with reference to SEQ ID NO: 2 and whereinthe variant demonstrates any one of

-   -   a) increased (thermo)stability; or    -   b) increased specific activity; or    -   c) increased sucrose tolerance; or    -   d) increased stability/activity at different pH range; or    -   e) change in product spectrum (defined as ration of one product        over another); or    -   f) increased activity on raw starch; or    -   g) altered temperature optimum; or    -   h) alter substrate specificity; or    -   i) increased productivity in the production of the alpha-amylase        variant;        as compared with a reference polypeptide having alpha-amylase        activity.        In an embodiment according to the invention there is provided a        variant polypeptide having alpha-amylase activity, wherein the        variant has an amino acid sequence which, when aligned with the        alpha-amylase comprising the sequence set out in SEQ ID NO: 2,        comprises at least one substitution of an amino acid residue        corresponding to any of amino acids

4, 6, 13, 14, 15, 16, 20, 45, 47, 51, 54, 61, 68, 69, 70, 71, 72, 73,74, 75, 77, 78, 80, 82, 87, 88, 94, 95, 100, 103, 104, 117, 124, 125,126, 128, 130, 133, 134, 136, 143, 144, 146, 168, 174, 177, 178, 183,186, 188, 189, 190, 194, 195, 199, 200, 201, 204, 207, 210, 214, 217,219, 222, 225, 227, 233, 234, 235, 236, 240, 251, 252, 254, 258, 259,260, 261, 262, 263, 264, 266, 267, 269, 271, 273, 281, 282, 283, 284,286, 288, 299, 322, 323, 325, 327, 328, 331, 334, 350, 356, 358, 367,370, 371, 374, 377, 378, 388, 391, 414, 421, 422, 445, 450, 467, 488,505, 536, 548, 583, 588, 603, 637, 648, 651, 652, 660, 676, 677,

said positions being defined with reference to SEQ ID NO: 2 and whereinthe variant demonstrates any one of

-   -   a) increased (thermo)stability; or    -   b) increased specific activity; or    -   c) increased sucrose tolerance; or    -   d) increased stability/activity at different pH range; or    -   e) change in product spectrum (defined as ration of one product        over another); or    -   f) increased activity on raw starch; or    -   g) altered temperature optimum; or    -   h) alter substrate specificity; or    -   i) increased productivity in the production of the alpha-amylase        variant;        as compared with a reference polypeptide having alpha-amylase        activity.        In an aspect the alpha-amylase variant according to the        invention, is an alpha-amylase variant having at least 80%        identity, in an aspect at least 85% identity, in an aspect at        least 90% identity, in an aspect at least 95% identity, in an        aspect at least 98% identity, in an aspect at least 99%        identity, in an aspect at least 99.5% identity, with the        polypeptide sequence as set out in SEQ ID NO: 2,

wherein at least one amino acid residue of the variant is substituted ata position selected from the group consisting of 4, 6, 13, 14, 15, 16,20, 45, 47, 51, 54, 61, 68, 69, 70, 71, 72, 73, 74, 75, 77, 78, 80, 82,87, 88, 94, 95, 100, 103, 104, 117, 124, 125, 126, 128, 130, 133, 134,136, 143, 144, 146, 168, 174, 177, 178, 183, 186, 188, 189, 190, 194,195, 199, 200, 201, 204, 207, 210, 214, 217, 219, 222, 225, 227, 233,234, 235, 236, 240, 251, 252, 254, 258, 259, 260, 261, 262, 263, 264,266, 267, 269, 271, 273, 281, 282, 283, 284, 286, 288, 299, 322, 323,325, 327, 328, 331, 334, 350, 356, 358, 367, 370, 371, 374, 377, 378,388, 391, 414, 421, 422, 445, 450, 467, 488, 505, 536, 548, 583, 588,603, 637, 648, 651, 652, 660, 676, 677,

said positions being defined with reference to SEQ ID NO: 2;

and wherein the variant has a specific activity which is higher at atleast one pH, preferably a pH between 4 and 8, than that of thepolypeptide as set out in SEQ ID NO: 2 measured at the same pH and/orwherein the variant has a pH optimum which is higher than that of thepolypeptide as set out in SEQ ID NO: 2.

In one embodiment an alpha-amylase variant according to the inventionmay have a pH optimum which is altered compared to the parentpolypeptide

In one embodiment an alpha-amylase variant according to the inventionmay have a pH optimum which is higher than that of the parentpolypeptide having alpha-amylase activity or lower than such parentpolypeptide. In an aspect the pH optimum of the alpha-amylase variantprotein is higher than that of the parent polypeptide. Preferably theparent polypeptide is that according to SEQ ID NO: 2. In an aspect theparent polypeptide is the wild-type alpha-amylase from Alicyclobacilluspohliae (as disclosed in SEQ ID NO: 2). In an aspect an alpha-amylasevariant of the invention may be more alkaliphilic than such a wild-typeenzyme, i.e. may, for example, have a pH optimum of from pH 5 to pH 8,preferably from pH 6 to pH 7. Optionally a variant protein of theinvention may be more acidophilic than the wild type alpha-amylase.

In an aspect an alpha-amylase variant according to the invention mayhave a pH, which is higher than the pH optimum and at which 50% of thealpha-amylase activity is still present, (hereafter indicated asalkaline pH), which is higher than that of the parent alpha-amylase.When the parent polypeptide having alpha-amylase activity is thataccording to SEQ ID NO: 2 the variant protein may have an alkaline pH atwhich 50% of the activity is observed which is at least 6.9, preferably,at least 7.0, at least 7.5, preferably at least 8.

In an aspect an alpha-amylase variant according to the invention mayhave a pH, which is lower than the pH optimum and at which 50% of thealpha-amylase activity is still present, (hereafter indicated as acidicpH), which is lower than that of the parent alpha-amylase. In an aspectthe alpha-amylase variant according to the invention may have an acidicpH at which 50% of the alpha-amylase activity is observed which is atmost 3.8, in an aspect at most 3.7, in an aspect at most 3.6, in anaspect at most 3.5, in an aspect at most 3.3, in an aspect at most 3.0,in an aspect at most 2.7, in an aspect at most 2.5.

A variant which exhibits a property which is improved in relation to theparent polypeptide having alpha-amylase activity is one whichdemonstrates a measurable reduction or increase in the relevantproperty, typically such that the variant is more suited to use as setout below, for example in a method for the production of a foodstuff.

In an aspect an alpha-amylase variant according to the invention mayhave a specific activity which is higher than that of the parentpolypeptide measured at the same pH. With specific activity of a variantprotein it is herewith intended the alpha-amylase activity of thealpha-amylase variant measured in units/mg of pure protein. Preferablythe specific activity of the alpha-amylase variant according to theinvention is higher at at least one pH, preferably a pH between 4 and 8,than that of the parent polypeptide measured at the same pH.

In an embodiment of the alpha-amylase variant according to the inventioncomprises an amino acid sequence which, when aligned with the sequenceset out in SEQ ID NO: 2, comprises at least one substitution of an aminoacid residue corresponding to any of amino acids

-   1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19,    20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,    37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 71,    72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,    89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,    105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,    118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,    131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,    144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,    157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,    170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,    183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,    196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,    209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,    222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,    235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247,    248, 249, 250, 251, 251, 253, 254, 255, 256, 257, 258, 259, 260,    261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273,    274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,    287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299,    300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312,    313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325,    326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,    339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351,    351, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364,    365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377,    378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390,    391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403,    404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416,    417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429,    430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442,    443, 444, 445, 446, 447, 448, 449, 450, 451, 451, 453, 454, 455,    456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468,    469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481,    482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494,    495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507,    508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520,    521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533,    534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546,    547, 548, 549, 550, 551, 551, 553, 554, 555, 556, 557, 558, 559,    560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572,    573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585,    586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598,    599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611,    612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624,    625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637,    638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650,    651, 651, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663,    664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676,    677, 678, 679, 680, 681, 682, 683, 684, 685, 686,

said positions being defined with reference to SEQ ID NO: 2 and whereinthe variant demonstrates any one of

-   -   j) a pH optimum at a pH lower than 5.0, in an aspect lower than        4.9, in an aspect lower than 4.8, in an aspect lower than 4.7,        in an aspect lower than 4.6, in an aspect lower than 4.5, in an        aspect lower than 4.4, in an aspect lower than 4.3, in an aspect        lower than 4.2; or    -   k) a pH optimum in a range of from 4.0 to 4.8, in an aspect of        from 4.2 to 4.7; or    -   l) an alkaline pH at which 50% of the activity is observed as        compared to the alpha-amylase activity at the pH optimum which        is set at 100%, which is at least 6.9, in an aspect at least        7.0, in an aspect at least 7.5, in an aspect at least 8; or    -   m) an acidic pH at which 50% of the alpha-amylase activity is        observed as compared to the alpha-amylase activity at the pH        optimum which is set at 100%, which is at most 3.8, in an aspect        at most 3.7, in an aspect at most 3.6, in an aspect at most 3.5,        in an aspect at most 3.3, in an aspect at most 3.0, in an aspect        at most 2.7, in an aspect at most 2.5.        In an embodiment according to the invention there is provided a        variant polypeptide having alpha-amylase activity, wherein the        variant has an amino acid sequence which, when aligned with the        alpha-amylase comprising the sequence set out in SEQ ID NO: 2,        comprises at least one substitution of an amino acid residue        corresponding to any of amino acids

4, 6, 13, 14, 15, 16, 20, 45, 47, 51, 54, 61, 68, 69, 70, 71, 72, 73,74, 75, 77, 78, 80, 82, 87, 88, 94, 95, 100, 103, 104, 117, 124, 125,126, 128, 130, 133, 134, 136, 143, 144, 146, 168, 174, 177, 178, 183,186, 188, 189, 190, 194, 195, 199, 200, 201, 204, 207, 210, 214, 217,219, 222, 225, 227, 233, 234, 235, 236, 240, 251, 252, 254, 258, 259,260, 261, 262, 263, 264, 266, 267, 269, 271, 273, 281, 282, 283, 284,286, 288, 299, 322, 323, 325, 327, 328, 331, 334, 350, 356, 358, 367,370, 371, 374, 377, 378, 388, 391, 414, 421, 422, 445, 450, 467, 488,505, 536, 548, 583, 588, 603, 637, 648, 651, 652, 660, 676, 677,

said positions being defined with reference to SEQ ID NO: 2 and whereinthe variant demonstrates any one of

-   -   j) a pH optimum at a pH lower than 5.0, in an aspect lower than        4.9, in an aspect lower than 4.8, in an aspect lower than 4.7,        in an aspect lower than 4.6, in an aspect lower than 4.5, in an        aspect lower than 4.4, in an aspect lower than 4.3, in an aspect        lower than 4.2; or    -   k) a pH optimum in a range of from 4.0 to 4.8, in an aspect of        from 4.2 to 4.7; or    -   l) an alkaline pH at which 50% of the activity is observed as        compared to the alpha-amylase activity at the pH optimum which        is set at 100%, which is at least 6.9, in an aspect at least        7.0, in an aspect at least 7.5, in an aspect at least 8; or    -   m) an acidic pH at which 50% of the alpha-amylase activity is        observed as compared to the alpha-amylase activity at the pH        optimum which is set at 100%, which is at most 3.8, in an aspect        at most 3.7, in an aspect at most 3.6, in an aspect at most 3.5,        in an aspect at most 3.3, in an aspect at most 3.0, in an aspect        at most 2.7, in an aspect at most 2.5.        The pH optimum may be determined by determining the dependence        of the enzyme activity of the alpha-amylase variant according to        the invention on pH. The alkaline pH and/or acidic pH may be        determined by determining the dependence of the enzyme activity        of the alpha-amylase variant according to the invention on pH.        Such dependence may be determined by applying the NBAU assay as        described herein using a reaction mixture in which the pH is        adjusted to different pH values. Suitable different pH values        include, but are not limited to, any one of 2.5, 2.7, 3.0, 3.3,        3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.5, 4.6, 4.7, 4.8,        4.9, 5.0, 5.1, 5.2, 5.3, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2,        6.3, 6.4, 6.5, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 8.0,        8.5.        This way the relative activity at different pH may be        determined. The pH optimum is defined as the pH at which the        enzyme is having the highest relative alpha-amylase activity,        i.e. 100%. The alkaline pH is the pH which is higher than the pH        optimum and at which 50% of the alpha-amylase activity is still        present as compared to the alpha-amylase activity at the pH        optimum which is set at 100%. The acidic pH is the pH which is        lower than the pH optimum and at which 50% of the alpha-amylase        activity is still present as compared to the alpha-amylase        activity at the pH optimum which is set at 100%.        In an embodiment the alpha-amylase variant according to the        invention has a Cys amino acid at any one of both of 4 and 505,        77 and 88, 78 and 134, 82 and 144, 207 and 676, 207 and 676, 207        and 677, 240 and 583, 488 and 467, 536 and 548, 583 and 236, 588        and 651 or 677 and 204, said positions being defined with        reference to SEQ ID NO: 2 and wherein the variant has one or        more altered properties as compared with a reference polypeptide        having alpha-amylase activity.

In an embodiment the alpha-amylase variant according to the inventionthe reference polypeptide is the alpha-amylase of SEQ ID NO: 2

In an embodiment the alpha-amylase variant according to the inventioncomprises an amino acid sequence which, when aligned with the sequenceset out in SEQ ID NO: 2, comprises at least one substitution of an aminoacid residue corresponding to any of amino acids

4, 6, 13, 14, 15, 16, 20, 45, 47, 51, 54, 61, 68, 69, 70, 71, 72, 73,74, 75, 77, 78, 80, 82, 87, 88, 94, 95, 100, 103, 104, 117, 124, 125,126, 128, 130, 133, 134, 136, 143, 144, 146, 168, 174, 177, 178, 183,186, 188, 189, 190, 194, 195, 199, 200, 201, 204, 207, 210, 214, 217,219, 222, 225, 227, 233, 234, 235, 236, 240, 251, 252, 254, 258, 259,260, 261, 262, 263, 264, 266, 267, 269, 271, 273, 281, 282, 283, 284,286, 288, 299, 322, 323, 325, 327, 328, 331, 334, 350, 356, 358, 367,370, 371, 374, 377, 378, 388, 391, 414, 421, 422, 445, 450, 467, 488,505, 536, 548, 583, 588, 603, 637, 648, 651, 652, 660, 676, 677,

said positions being defined with reference to SEQ ID NO: 2 and whereinthe variant demonstrates any one of

-   -   a) increased (thermo)stability; or    -   b) increased specific activity; or    -   c) increased sucrose tolerance; or    -   d) increased stability/activity at different pH range; or    -   e) change in product spectrum (defined as ration of one product        over another); or    -   f) increased activity on raw starch; or    -   g) altered temperature optimum; or    -   h) alter substrate specificity; or    -   i) increased productivity in the production of the alpha-amylase        variant;

as compared with a reference polypeptide having alpha-amylase activity;or wherein the variant demonstrates any one of

-   -   j) a pH optimum at a pH lower than 5.0; or    -   k) a pH optimum in a range of from 4.0 to 4.8; or    -   l) an alkaline pH at which 50% of the activity is observed as        compared to the alpha-amylase activity at the pH optimum which        is set at 100%, which is at least 6.9; or    -   m) an acidic pH at which 50% of the alpha-amylase activity is        observed as compared to the alpha-amylase activity at the pH        optimum which is set at 100%, which is at most 3.8.

A preferred reference polypeptide suitable for use in the invention isthe polypeptide having the sequence set out in SEQ ID NO: 2 or having atleast 80% homology with SEQ ID NO: 2, for example at least 85% homologywith SEQ ID NO: 2, such as a least 85% homology with SEQ ID NO: 2, suchas at least 90% homology with SEQ ID NO: 2, for example at least 95%, atleast 98%, at least 99% or at least 99.5% homology with SEQ ID NO: 2.

The amino acid residues in a variant alpha-amylase of the invention thatmay be substituted with comparison with the sequence set out in SEQ IDNO: 2 are those which correspond to positions

-   1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19,    20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,    37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 71,    72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,    89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,    105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,    118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,    131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,    144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,    157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,    170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,    183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,    196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,    209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,    222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,    235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247,    248, 249, 250, 251, 251, 253, 254, 255, 256, 257, 258, 259, 260,    261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273,    274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,    287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299,    300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312,    313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325,    326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,    339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351,    351, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364,    365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377,    378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390,    391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403,    404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416,    417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429,    430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442,    443, 444, 445, 446, 447, 448, 449, 450, 451, 451, 453, 454, 455,    456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468,    469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481,    482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494,    495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507,    508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520,    521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533,    534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546,    547, 548, 549, 550, 551, 551, 553, 554, 555, 556, 557, 558, 559,    560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572,    573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585,    586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598,    599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611,    612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624,    625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637,    638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650,    651, 651, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663,    664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676,    677, 678, 679, 680, 681, 682, 683, 684, 685, 686,

as defined in relation to the sequence of SEQ ID NO: 2.

In an embodiment the amino acid residues in a variant alpha-amylase ofthe invention that may be substituted with comparison with the sequenceset out in SEQ ID NO: 2 are those which correspond to positions

4, 6, 13, 14, 15, 16, 20, 45, 47, 51, 54, 61, 68, 69, 70, 71, 72, 73,74, 75, 77, 78, 80, 82, 87, 88, 94, 95, 100, 103, 104, 117, 124, 125,126, 128, 130, 133, 134, 136, 143, 144, 146, 168, 174, 177, 178, 183,186, 188, 189, 190, 194, 195, 199, 200, 201, 204, 207, 210, 214, 217,219, 222, 225, 227, 233, 234, 235, 236, 240, 251, 252, 254, 258, 259,260, 261, 262, 263, 264, 266, 267, 269, 271, 273, 281, 282, 283, 284,286, 288, 299, 322, 323, 325, 327, 328, 331, 334, 350, 356, 358, 367,370, 371, 374, 377, 378, 388, 391, 414, 421, 422, 445, 450, 467, 488,505, 536, 548, 583, 588, 603, 637, 648, 651, 652, 660, 676, 677,

as defined in relation to the sequence of SEQ ID NO: 2.

A variant alpha-amylase of the invention may comprises a substitution atone or more of the said positions, for example at two, three, four, atleast 5, at least 10, at least 15, at least 20, at least 25, at least30, at least 35, at least 40, at least 45 or at least 50 or at all ofthe said positions.

A variant alpha-amylase of the invention may comprise one or moresubstitutions as defined above. A “substitution” in this contextindicates that a position in the variant which corresponds to one of thepositions set out above in SEQ ID NO: 2 comprises an amino acid residuewhich does not appear at that position in the reference polypeptide (thereference polypeptide may be SEQ ID NO: 2).

Preferred substitutions are set out in the following Table 1 and Table 2(with the positions being defined in relation to the sequence set out inSEQ ID NO: 2).

A variant of the invention may be generated using any combination ofsubstitutions set out in Table 1 and/or Table 2.

TABLE 1 Preferred substitutions defined in relation to SEQ ID NO: 2Amino acids are depicted according to the single letter annotation #Substitution  01 V6F   2 I14V   3 I15F   4 I15V   5 I16T   6 M45L   7W47F   8 L51W   9 V54L  10 V54I  11 L61F  12 I69L  13 L71I  14 P73Q  15V74P  16 L75V  17 L75F  18 L78I  19 L78V  20 T80A  21 T87V  22 G88A  23G88S  24 T94A  25 T94P  26 R95K  27 I100T  28 H103Y  29 F104Y  30 V124I 31 I125V  32 V126I  33 V129A  34 V129T  35 P130V  36 S133T  37 T134S 38 F136A  39 F143Y  40 G146N  41 F168Y  42 I174L  43 I174V  44 W177F 45 D178N  46 A183S  47 K186R  48 K186Q  49 F188I  50 T189Y  51 D190N 52 F194Y  53 S195T  54 S195A  55 S195N  56 L199F  57 S200N  58 Q201H 59 L210F  60 A214I  61 L217Y  62 L217W  63 A219D  64 A222V  65 A222I 66 I227V  67 F233Y  68 F233M  69 N234P  70 S235L  71 I251V  72 V254F 73 Y258F  74 G259L  75 D260G  76 P262S  77 G263A  78 A264S  79 N266S 80 H267N  81 E269D  82 V271T  83 V271I  84 Y273F  85 V279M  86 V281L 87 L286F  88 T288S  89 T288N  90 T288Q  91 T323N  92 I325F  93 N327S 94 H328Q  95 S331D  96 I350T  97 T356V  98 S358A  99 M367L 100 G370N101 N371G 102 Y374D 103 G377A 104 M378K 105 W414Y 106 I421V 107 Y422F108 I445V 109 T603S 110 T603V 111 Y637F 112 Y637I 113 Q648E 114 T450S115 F652I 116 I660V 117 Q13E 118 Y20L 119 Y20V 120 T68A 121 T68S 122T68G 123 W70Y 124 S72T 125 A117V 126 A117C 127 F128Y 128 F128I 129 F128L130 L225I 131 L225F 132 L225W 133 F252Y 134 F252L 135 L282W 136 L282F137 L282I 138 L282M 139 L282T 140 Q299S 141 L334K 142 L334Y 143 L334Q144 L334H 145 L282M 146 D283N 147 D283S 148 F284Y 149 F284I 150 F284M151 F284W 152 F284L 153 I322V 154 I322F 155 I322P 156 A388L 157 A388S158 E391V 159 D261G 160 A4C-A505C 161 N77C-G88C 162 L78C-T134C 163A82C-A144C 164 A207C-A676C 165 A207C-T677C 166 S240C-S583C 167S488C-G467C 168 A536C-V548C 169 S583C-G236C 170 V588C-F651C 171T677C-G204C

TABLE 2 possible substitutions, position in  reference to SEQ ID NO: 2Amino acids are depicted  according to the single letter annotationChange to amino acid (multiple  options from which a selection  positionmay be made are separated by /) 13 S/T/A/V/L/I/F/M 15 T/S/V/L/D 17 Q/E18 K 26 S/T/A/V/L/I 30 D/M/L/A/V/I/E/Q 32 D/E/N/Q 35 Q 40 R 44 R/S/T/Q/N45 K 51 W 73 Q 74 P 75 F/Y 76 E 77 S/T/A/V/L/I 78 I 79 E/Y 86S/T/A/V/L/I/Q/G/K 87 N/Q/S 88 A/S/T 89 W/F/H 90 W/F/Y/R/K/N/Q/M 91T/S/V/N 93 S/G/V/T/M/E/Y/F 94 V/I/L 95 L/A/V/I/E/Q 99 T/S/V/L 100T/S/D/N/E/Q 103 Y/V/I/L/F/Y/N/Q/D/E 114 V/I/L 119 T/S 120 S/T/A/V/L/I125 L/M/F/Y/W 126 I/L 127 N/L 129 T/G/V 131 D 131 S/T/A/V/L/I 134A/V/I/L 141 P 142 A 148 D/N/E/Q/S/T/R/K 152 T/S/V/L 157 V/I/M/F/Y/W 163Y 169 N/D/E/Q 171 Y/D/S/T 172 S/D/N/V 174 E/Q 176 S/T/A/V/L/I 178L/M/T/V 187 S/T/A/V/L/I 188 E/K/H/I/L/G/T/V/ 189 M 190 E/Q/G 190 G 192S/D/N/G/T/Q/R 194 S/L/Y 196 F 198 G 201 E 201 S/T/A/V/L/I/F/M 203D/S/T/A/V/L/I 217 I/L/M/F/Y/W 220 Y/L/M 225 S 227 V 229 T/G/V 230 G 231R/L/M 234 S/T/A/V/L/I/P 235 A/V/I/L/M/F/Y/W 236 I/L/M/F/Y/W 247S/T/A/V/L/I/F/M 249 P 252 L 254 I/L/F 258 D/K/R/F/N/W/L/M/T/V 259 A/H/Y260 L/M/T/V 261 G 264 Y/Q/F/A/V 266 S/T/A/V/L/I/Y 268 R/K/P 275S/T/A/V/L/I 279 M/I/L/F/P 280 S/T/A/V/L/I 281 I/L/M/F/Y/W/T/Q 284K/H/D/E/Y 285 R/K 286 F 287 S/T/A/V/L/I 288 Y/Q/F/A/V/P/E/K/R 289 I/L/R290 M/L/F/V 297 N/D/Q/E 299 L/T/S 305 K/R 308 I/L/M/F/Y/W 316 N/D 320S/T/A/V/L/I 321 I/M/F/Y/W/Q 325 L/M/F/Y/W 326 E/Q 327D/K/R/F/N/S/T/A/V/L/I 330 L/F/I/D/E/K 341 R/K 342 S/T/A/V/L/I/P 343M/F/Y/W 344 E/Q/N/D/Y 349 W/Y 353 V/I/L 359 L/M/F/Y/W 365 S/T/A/V/L/I370 N/L 371 D/E/G/K/S/T/A/V/L/I/R/F/Y/Q 372 E/Q/S/T/A/V/N 375S/T/A/V/L/I 378 R/K 381 S/D/N 389 Y 397 P 401 S/T/A/V/L/I 403 P 405M/L/Y/F/W 425 E 436 S/T/A/V/L/I 442 P 446 A 448 Y 449 Y 452 M/Y/F/W 454D/S/T/A/V/L/I 468 D/S/T/A/V/L/I 469 R 470 M/L/F 474 D/S/T/A/V/L/I 479 P483 S/D/N 486 Q/E 493 P 494 P 495 P 496 P 497 P 498 P 500S/T/A/V/L/I/F/M/P 507 S/T/A/V/L/I 509 A/V/I/L/M/S/T/D/N 510 R/K 513S/T/A/V/L/I 515 I/L 520 R 526 D/S/T/A/V/L/I 555 P 557 Q/E/N/D 564 S/D/N573 N/D 575 S/T/A/V/L/I 578 G 581 S/T/A/V/L/I/F/M 583 V/I/L 586 S/D/N589 S/D/N/Q 595 I/L 621 S/T/A/V/L/I 624 S/T/A/V/L/I/F/M 625A/V/I/L/M/F/Y/W 627 M/F/Y 628 M/I/F/Y/W 629 N/D/E/Q 636 Y 642 Q 645 T664 D/S/T/A/V/L/I 670 V/I/L/M/F/Y/W 681 D/N/E/Q/S

A variant alpha-amylase of the invention may also comprise additionalmodifications in comparison to the parent at positions other than thosespecified above, for example, one or more additional substitutions,additions or deletions. A variant of the invention may comprise acombination of different types of modification of this sort. A variantmay comprise one, two, three, four, least 5, at least 10, at least 15,at least 20, at least 25, at least 30 or more such modifications (whichmay all be of the same type or may be different types of modification).Typically, the additional modifications may be substitutions.

A variant according to the invention may have at least 80% homology withthe reference alpha-amylase polypeptide, such as the alpha-amylase ofSEQ ID NO: 2, for example at least 85% homology with the parentpolypeptide, such as 90% homology with the parent polypeptide, at least95% homology with the parent polypeptide, at least 98% homology with theparent polypeptide, at least 99% homology with the parent polypeptide orat least 99.5% homology with the parent polypeptide.

A variant of the invention will typically retain alpha-amylase activity.That is to say, a variant of the invention will typically be capable ofalpha amylase activity. Alpha-amylase activity can suitably bedetermined using the Ceralpha® procedure, which is recommended by theAmerican Association of Cereal Chemists (AACC).

A variant of the invention will typically be a starch degrading enzyme.

Preferably, a variant of the invention will typically exhibit improvedproperties in comparison with the reference alpha-amylase polypeptidefrom which it is derived. Such an improved property will typically beone which is relevant if the variant were to be used as set out herein,for example in a method for preparing a baked product.

A variant which exhibits a property which is improved in relation to thereference alpha-amylase is one which demonstrates a measurable reductionor increase in the relevant property, typically such that the variant ismore suited to use as set out herein, for example in a method for theproduction of a baked product.

The property may thus be decreased by at least 10%, at least 20%, atleast 30%, at least 40% at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95% or at least 99%. Alternatively,the property may be increased by at least 10%, at least 25%, at least50%, at least 100%, at least, 200%, at least 500% or at least 1000%. Thepercentage decrease or increase in this context represents thepercentage decrease or increase in comparison to the referencealpha-amylase polypeptide. It is well known to the skilled person howsuch percentage changes may be measured—it is a comparison of theactivity of the reference alpha-amylase and the variant alpha-amylase.

The variants described herein are collectively comprised in the terms “apolypeptide according to the invention” or “a variant according to theinvention”.

The terms “peptide” and “oligopeptide” are considered synonymous (as iscommonly recognized) and each term can be used interchangeably as thecontext requires to indicate a chain of at least two amino acids coupledby peptidyl linkages. The word “polypeptide” is used herein for chainscontaining more than about seven amino acid residues. All oligopeptideand polypeptide formulas or sequences herein are written from left toright and in the direction from amino terminus to carboxy terminus. Theone-letter code of amino acids used herein is commonly known in the artand can be found in Sambrook, et al. (Molecular Cloning: A LaboratoryManual, 2nd, ed. Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989).

A polypeptide of the invention may be in isolated form, such assubstantially isolated form. By “isolated” polypeptide or protein isintended a polypeptide or protein removed from its native environment.For example, recombinantly produced polypeptides and proteins expressedin host cells are considered isolated for the purpose of the inventionas are recombinant polypeptides which have been substantially purifiedby any suitable technique. A polypeptide variant according to theinvention can be recovered and purified from recombinant cell culturesby methods known in the art.

Polypeptides of the present invention include products of chemicalsynthetic procedures, and products produced by recombinant techniquesfrom a prokaryotic or eukaryotic host, including, for example,bacterial, yeast, higher plant, insect and mammalian cells. Dependingupon the host employed in a recombinant production procedure, thepolypeptides of the present invention may be glycosylated or may benon-glycosylated. In addition, polypeptides of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes.

The invention also features biologically active fragments of thepolypeptide variants according to the invention. Such fragments areconsidered to be encompassed within the term “a variant of theinvention”.

Biologically active fragments of a polypeptide variant of the inventioninclude polypeptides comprising amino acid sequences sufficientlyidentical to or derived from the amino acid sequence of a variantprotein of the invention which include fewer amino acids than the fulllength protein but which exhibit at least one biological activity of thecorresponding full-length protein. Typically, biologically activefragments comprise a domain or motif with at least one activity of avariant protein of the invention. A biologically active fragment of aprotein of the invention can be a polypeptide which is, for example, 10,25, 50, 100 or more amino acids in length. Moreover, other biologicallyactive portions, in which other regions of the protein are deleted, canbe prepared by recombinant techniques and evaluated for one or more ofthe biological activities of the native form of a polypeptide of theinvention.

Typically, a protein fragment of the invention will comprise one or moreof the substitutions defined herein.

The invention also features nucleic acid fragments which encode theabove biologically active fragments (which biologically active fragmentsare themselves variants of the invention).

As set out above, the present invention provides polynucleotidesencoding the variant polypeptides of the invention. The invention alsorelates to an isolated polynucleotide encoding at least one functionaldomain of a polypeptide variant of the invention. Typically, such adomain will comprise one or more of the substitutions described herein.

In one embodiment of the invention, the nucleic acid sequence accordingto the invention encodes a polypeptide, wherein the polypeptide is avariant comprising an amino acid sequence that has one or moretruncation(s), and/or at least one substitution, deletion and/orinsertion of an amino acid as compared to the parent alpha-amylase. Sucha polypeptide will, however, typically comprise one or more of thesubstitutions described herein.

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules which include an open reading frame encoding a variant asdescribed herein. A gene may include coding sequences, non-codingsequences, introns and regulatory sequences. That is to say, a “gene”,as used herein, may refer to an isolated nucleic acid molecule asdefined herein. Accordingly, the term “gene”, in the context of thepresent application, does not refer only to naturally-occurringsequences.

A nucleic acid molecule of the present invention can be generated usingstandard molecular biology techniques well known to those skilled in theart taken in combination with the sequence information provided herein.

A nucleic acid molecule of the present invention can be adaptedaccording to the method described in patent application US090286280.

For example, using standard synthetic techniques, the required nucleicacid molecule may be synthesized de novo. Such a synthetic process willtypically be an automated process.

Alternatively, a nucleic acid molecule of the invention may be generatedby use of site-directed mutagenesis of an existing nucleic acidmolecule, for example a wild-type nucleic acid molecule. Site-directedmutagenesis may be carried out using a number of techniques well know tothose skilled in the art.

In one such method, mentioned here merely by way of example, PCR iscarried out on a plasmid template using oligonucleotide “primers”encoding the desired substitution. As the primers are the ends ofnewly-synthesized strands, should there be a mis-match during the firstcycle in binding the template DNA strand, after that first round, theprimer-based strand (containing the mutation) would be at equalconcentration to the original template. After successive cycles, itwould exponentially grow, and after 25, would outnumber the original,unmutated strand in the region of 8 million: 1, resulting in a nearlyhomogeneous solution of mutated amplified fragments. The template DNAmay then be eliminated by enzymatic digestion with, for example using arestriction enzyme which cleaves only methylated DNA, such as Dpn1. Thetemplate, which is derived from an alkaline lysis plasmid preparationand therefore is methylated, is destroyed in this step, but the mutatedplasmid is preserved because it was generated in vitro and isunmethylated as a result.

In such a method more than one mutation (encoding a substitution asdescribed herein) may be introduced into a nucleic acid molecule in asingle PCR reaction, for example by using one or more oligonucleotides,each comprising one or more mis-matches. Alternatively, more than onemutation may be introduced into a nucleic acid molecule by carrying outmore than one PCR reaction, each reaction introducing one or moremutations, so that altered nucleic acids are introduced into the nucleicacid in a sequential, iterative fashion.

A nucleic acid of the invention can be generated using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriate mis-matchedoligonucleotide primers according to the site-directed mutagenesistechnique described above. A nucleic acid molecule derived in this waycan be cloned into an appropriate vector and characterized by DNAsequence analysis.

A nucleic acid sequence of the invention may comprise one or moredeletions, i.e. gaps, in comparison to the parent alpha-amylase. Suchdeletions/gaps may also be generated using site-directed mutagenesisusing appropriate oligonucleotides. Techniques for generating suchdeletions are well known to those skilled in the art.

Furthermore, oligonucleotides corresponding to or hybridizable tonucleotide sequences according to the invention can be prepared bystandard synthetic techniques, e.g., using an automated DNA synthesizer.

Also, complementary nucleic acid molecules are included in the presentinvention. A nucleic acid molecule which is complementary to anothernucleotide sequence is one which is sufficiently complementary to theother nucleotide sequence such that it can hybridize to the othernucleotide sequence thereby forming a stable duplex.

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode a variant of the invention, or a biologically activefragment or domain thereof, as well as nucleic acid molecules sufficientfor use as hybridization probes to identify nucleic acid moleculesencoding a polypeptide of the invention and fragments of such nucleicacid molecules suitable for use as PCR primers for the amplification ormutation of nucleic acid molecules, such as for the preparation ofnucleic acid molecules of the invention.

An “isolated polynucleotide” or “isolated nucleic acid” is a DNA or RNAthat is not immediately contiguous with both of the coding sequenceswith which it is immediately contiguous (one on the 5′ end and one onthe 3′ end) in the naturally occurring genome of the organism from whichit is derived. Thus, in one embodiment, an isolated nucleic acidincludes some or all of the 5′ non-coding (e.g., promotor) sequencesthat are immediately contiguous to the coding sequence. The termtherefore includes, for example, a recombinant DNA that is incorporatedinto a vector, into an autonomously replicating plasmid or virus, orinto the genomic DNA of a prokaryote or eukaryote, or which exists as aseparate molecule (e.g., a cDNA or a genomic DNA fragment produced byPCR or restriction endonuclease treatment) independent of othersequences. It also includes a recombinant DNA that is part of a hybridgene encoding an additional polypeptide that is substantially free ofcellular material, viral material, or culture medium (when produced byrecombinant DNA techniques), or chemical precursors or other chemicals(when chemically synthesized). Moreover, an “isolated nucleic acidfragment” is a nucleic acid fragment that is not naturally occurring asa fragment and would not be found in the natural state.

As used herein, the terms “polynucleotide” or “nucleic acid molecule”are intended to include DNA molecules (e.g., cDNA or genomic DNA) andRNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated usingnucleotide analogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA. The nucleic acidmay be synthesized using oligonucleotide analogs or derivatives (e.g.,inosine or phosphorothioate nucleotides). Such oligonucleotides can beused, for example, to prepare nucleic acids that have alteredbase-pairing abilities or increased resistance to nucleases.

Another embodiment of the invention provides an isolated nucleic acidmolecule which is antisense to a nucleic acid molecule of the invention.

The terms “homology” or “percent identity” are used interchangeablyherein. For the purpose of this invention, it is defined here that inorder to determine the percent identity of two amino acid sequences ortwo nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in the sequence of afirst amino acid or nucleic acid for optimal alignment with a secondamino or nucleic acid sequence). The amino acid or nucleotide residuesat corresponding amino acid or nucleotide positions are then compared.When a position in the first sequence is occupied by the same amino acidor nucleotide residue as the corresponding position in the secondsequence, then the molecules are identical at that position. The percentidentity between the two sequences is a function of the number ofidentical positions shared by the sequences (i.e., % identity=number ofidentical positions/total number of positions (i.e. overlappingpositions)×100). Preferably, the two sequences are the same length.

A sequence comparison may be carried out over the entire lengths of thetwo sequences being compared or over fragment of the two sequences.Typically, the comparison will be carried out over the full length ofthe two sequences being compared. However, sequence identity may becarried out over a region of, for example, twenty, fifty, one hundred ormore contiguous amino acid residues.

The skilled person will be aware of the fact that several differentcomputer programs are available to determine the homology between twosequences. For instance, a comparison of sequences and determination ofpercent identity between two sequences can be accomplished using amathematical algorithm. In a preferred embodiment, the percent identitybetween two amino acid or nucleic acid sequences is determined using theNeedleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithmwhich has been incorporated into the GAP program in the Accelrys GCGsoftware package (available at www.accelrys.com/products/gcg/), usingeither a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16,14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. Theskilled person will appreciate that all these different parameters willyield slightly different results but that the overall percentageidentity of two sequences is not significantly altered when usingdifferent algorithms.

The protein sequences or nucleic acid sequences of the present inventioncan further be used as a “query sequence” to perform a search againstpublic databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the BLASTN andBLASTP programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST protein searches can be performed with the BLASTPprogram, score =50, wordlength =3 to obtain amino acid sequenceshomologous to protein molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the defaultparameters of the respective programs (e.g., BLASTP and BLASTN) can beused. See the homepage of the National Center for BiotechnologyInformation at www.ncbi.nlm.nih.gov/.

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a variantalpha-amylase polypeptide of the invention.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments can beligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. The terms “plasmid” and “vector” can be usedinterchangeably herein as the plasmid is the most commonly used form ofvector. However, the invention is intended to include such other formsof expression vectors, such as viral vectors (e.g., replicationdefective retroviruses, adenoviruses and adeno-associated viruses),which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorincludes one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operatively linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signal). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cells and those which directexpression of the nucleotide sequence only in a certain host cell (e.g.tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, encoded by nucleic acids as described herein (e.g.an alpha-amylase variant of SEQ ID NO: 2, for example a functionalequivalent or fragment, or a fusion protein comprising one or more ofsuch variants).

The recombinant expression vectors of the invention can be designed forexpression of variant proteins of the invention in prokaryotic oreukaryotic cells. For example, a variant protein of the invention can beexpressed in bacterial cells such as E. coli, insect cells (usingbaculovirus expression vectors) yeast cells or mammalian cells. Suitablehost cells are discussed further in Goeddel, Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example using T7 promoter regulatory sequencesand T7 polymerase.

Expression vectors useful in the present invention include chromosomal-,episomal- and virus-derived vectors e.g., vectors derived from bacterialplasmids, bacteriophage, yeast episome, yeast chromosomal elements,viruses such as baculoviruses, papova viruses, vaccinia viruses,adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses,and vectors derived from combinations thereof, such as those derivedfrom plasmid and bacteriophage genetic elements, such as cosmids andphagemids.

The DNA insert should be operatively linked to an appropriate promoter,such as the phage lambda PL promoter, the E. coli lac, trp and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name a few. Other suitable promoters will be known to theskilled person. In a specific embodiment, promoters are preferred thatare capable of directing a high expression level of alpha-amylase infilamentous fungi. Such promoters are known in the art. The expressionconstructs may contain sites for transcription initiation, termination,and, in the transcribed region, a ribosome binding site for translation.The coding portion of the mature transcripts expressed by the constructswill include a translation initiating AUG at the beginning and atermination codon appropriately positioned at the end of the polypeptideto be translated.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-percipitation, DEAE-dextran-mediated transfection,transduction, infection, lipofection, cationic lipidmediatedtransfection or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual, 2nd, ed. Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),Davis et al., Basic Methods in Molecular Biology (1986) and otherlaboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methatrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding a variant protein of the invention or can beintroduced on a separate vector. Cells stably transfected with theintroduced nucleic acid can be identified by drug selection (e.g. cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die).

Expression of proteins in prokaryotes is often carried out in E. coliwith vectors containing constitutive or inducible promoters directingthe expression of either fusion or non-fusion proteins. Fusion vectorsadd a number of amino acids to a protein encoded therein, e.g. to theamino terminus of the recombinant protein. Such fusion vectors typicallyserve three purposes: 1) to increase expression of recombinant protein;2) to increase the solubility of the recombinant protein; and 3) to aidin the purification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein.

As indicated, the expression vectors will preferably contain selectablemarkers. Such markers include dihydrofolate reductase or neomycinresistance for eukaryotic cell culture and tetracyline or ampicillinresistance for culturing in E. coli and other bacteria. Representativeexamples of appropriate host include bacterial cells, such as E. coli,Streptomyces Salmonella typhimurium and certain Bacillus species; fungalcells such as Aspergillus species, for example A. niger, A. oryzae andA. nidulans, such as yeast such as Kluyveromyces, for example K. lactisand/or Puchia, for example P. pastoris; insect cells such as DrosophilaS2 and Spodoptera Sf9; animal cells such as CHO, COS and Bowes melanoma;and plant cells. Appropriate culture mediums and conditions for theabove-described host cells are known in the art.

Vectors preferred for use in bacteria are for example disclosed inWO-A1-2004/074468, which are hereby enclosed by reference. Othersuitable vectors will be readily apparent to the skilled artisan.

Known bacterial promotors suitable for use in the present inventioninclude the promoters disclosed in WO-A1-2004/074468, which are herebyincorporated by reference.

Transcription of the DNA encoding a variant of the present invention byhigher eukaryotes may be increased by inserting an enhancer sequenceinto the vector. Enhancers are cis-acting elements of DNA, usually aboutfrom 10 to 300 bp that act to increase transcriptional activity of apromoter in a given host cell-type. Examples of enhancers include theSV40 enhancer, which is located on the late side of the replicationorigin at by 100 to 270, the cytomegalovirus early promoter enhancer,the polyoma enhancer on the late side of the replication origin, andadenovirus enhancers.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretation signal may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

A variant of the invention may be expressed in form such that it mayinclude additional heterologous functional regions, for examplesecretion signals. A variant of the invention may also comprise, forexample, a region of additional amino acids, particularly charged aminoacids, added to the N-terminus of the polypeptide for instance toimprove stability and persistence in the host cell, during purificationor during subsequent handling and storage. Also, peptide moieties may beadded to a variant of the invention to facilitate purification, forexample by the addition of histidine residues or a T7 tag.

The variants of the invention, such as proteins of the present inventionor functional equivalents thereof, e.g., biologically active portionsand fragments thereof, can be operatively linked to a non-variantpolypeptide (e.g., heterologous amino acid sequences) to form fusionproteins. A “non-variant polypeptide” in this context refers to apolypeptide having an amino acid sequence corresponding to a proteinwhich is not substantially homologous to a variant alpha-amylase of theinvention.

Within a fusion protein, the variant of the invention can correspond toa full length sequence or a biologically active fragment of apolypeptide of the invention. In a preferred embodiment, a fusionprotein of the invention comprises at least two biologically activeportions. Within the fusion protein, the term “operatively linked” isintended to indicate that the variant polypeptide and the non-variantpolypeptide are fused in-frame to each other. The non-variantpolypeptide can be fused to the N-terminus or C-terminus of the variantpolypeptide.

Expression and secretion of a variant alpha-amylase may be enhanced byexpressing the variant in the form of a fusion protein. In this context,a nucleic acid sequence may encode for a fusion protein comprisingpre-alpha-amylase or alpha-amylase. More specifically, the fusionpartner may be glucoamylase or a fragment thereof. In one embodiment thepre-alpha-amylase or alpha-amylase, or a fusion protein thereof, issecreted over the host cell membrane.

For example, in one embodiment, the fusion protein is a fusion proteinin which the variant sequence/s is/are fused to the C-terminus of theGST sequences. Such fusion proteins can facilitate the purification of arecombinant variant according to the invention. In another embodiment,the fusion protein is a variant of the invention containing aheterologous signal sequence at its N-terminus. In certain host cells(e.g., mammalian and yeast host cells), expression and/or secretion of avariant of the invention can be increased through use of a hetereologoussignal sequence.

In another example, the gp67 secretory sequence of the baculovirusenvelope protein can be used as a heterologous signal sequence (CurrentProtocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons,1992). Other examples of eukaryotic heterologous signal sequencesinclude the secretory sequences of melittin and human placental alkalinephosphatase (Stratagene; La Jolla, Calif.). In yet another example,useful prokarytic heterologous signal sequences include the phoAsecretory signal (Sambrook et al., supra) and the protein A secretorysignal (Pharmacia Biotech; Piscataway, N.J.).

A signal sequence can be used to facilitate secretion and isolation of avariant of the invention. Signal sequences are typically characterizedby a core of hydrophobic amino acids, which are generally cleaved fromthe mature protein during secretion in one or more cleavage events. Suchsignal peptides contain processing sites that allow cleavage of thesignal sequence from the mature proteins as they pass through thesecretory pathway. The signal sequence may direct secretion of thevariant, such as from a eukaryotic host into which the expression vectoris transformed, and the signal sequence may then be subsequently orconcurrently cleaved. The variant of the invention may then be readilypurified from the extracellular medium by known methods. Alternatively,the signal sequence can be linked to the variant of interest using asequence, which facilitates purification, such as with a GST domain.Thus, for instance, the sequence encoding the variant of the inventionmay be fused to a marker sequence, such as a sequence encoding apeptide, which facilitates purification of the fused variant of theinvention. In certain preferred embodiments of this aspect of theinvention, the marker sequence is a hexa-histidine peptide, such as thetag provided in a pQE vector (Qiagen, Inc.), among others, many of whichare commercially available. As described in Gentz et al, Proc. Natl.Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine providesfor convenient purification of the fusion protein. The HA tag is anotherpeptide useful for purification which corresponds to an epitope derivedof influenza hemaglutinin protein, which has been described by Wilson etal., Cell 37:767 (1984), for instance.

A fusion protein of the invention may be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent polypeptide sequences are ligated together in frame inaccordance with conventional techniques, for example by employingblunt-ended or stagger-ended termini for ligation, restriction enzymedigestion to provide for appropriate termini, filling-in of cohesiveends as appropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers, which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Current Protocols in Molecular Biology, eds. Ausubel et al.John Wiley & Sons: 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g, a GSTpolypeptide). A variant-encoding nucleic acid can be cloned into such anexpression vector such that the fusion moiety is linked in-frame to thesaid variant.

The terms “functional equivalents” and “functional variants” are usedinterchangeably herein. Functional equivalents according to theinvention are isolated DNA fragments that encode a polypeptide thatexhibits a particular function of a variant as defined herein.Functional equivalents therefore also encompass biologically activefragments and are themselves encompassed within the term “a variant” ofthe invention.

Preferably, a functional equivalent of the invention comprises one ormore of the substitutions described herein. However, a functionalequivalent may comprise one or more modifications in addition to thesubstitutions described above.

Functional nucleic acid equivalents may typically contain silentmutations or mutations that do not alter the biological function ofencoded polypeptide. Accordingly, the invention provides nucleic acidmolecules encoding a variant alpha-amylase protein that contains changesin amino acid residues that are not essential for a particularbiological activity. Such variant proteins differ in amino acid sequencefrom the parent alpha-amylase sequence from which they are derived yetretain at least one biological activity thereof, preferably they retainat least alpha-amylase activity. In one embodiment the isolated nucleicacid molecule comprises a nucleotide sequence encoding a protein,wherein the protein comprises a substantially homologous amino acidsequence of at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or more homologous to the reference amino acid sequence(for example that shown in SEQ ID NO: 2).

As defined herein, the term “substantially homologous” refers to a firstamino acid or nucleotide sequence which contains a sufficient or minimumnumber of identical or equivalent (e.g., with similar side chain) aminoacids or nucleotides to a second amino acid or nucleotide sequence suchthat the first and the second amino acid or nucleotide sequences have acommon domain. For example, amino acid or nucleotide sequences whichcontain a common domain having about 60%, preferably 65%, morepreferably 70%, even more preferably 75%, 80%, 85%, 90%, 95%, 96%, 97%,98% or 99% identity or more are defined herein as sufficientlyidentical.

The skilled person will recognise that changes can be introduced bymutation into the nucleotide sequences according to the inventionthereby leading to changes in the amino acid sequence of the resultingprotein without substantially altering the function of such a protein.

Accordingly, an alpha-amylase variant of the invention is preferably aprotein which comprises an amino acid sequence at least about 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous tothe reference amino acid sequence, for example that shown in SEQ ID NO:2, and typically also retains at least one functional activity of thereference polypeptide. Variants of the invention, for example functionalequivalents of a protein according to the invention, can also beidentified e.g. by screening combinatorial libraries of mutants, e.g.truncation mutants, of the protein of the invention for alpha-amylaseactivity. In one embodiment, a variegated library of variants isgenerated by combinatorial mutagenesis at the nucleic acid level. Avariegated library of variants can be produced by, for example,enzymatically ligating a mixture of synthetic oligonucleotides into genesequences such that a degenerate set of potential protein sequences isexpressible as individual polypeptides, or alternatively, as a set oflarger fusion proteins (e.g. for phage display). There are a variety ofmethods that can be used to produce libraries of potential variants ofthe polypeptides of the invention from a degenerate oligonucleotidesequence. Methods for synthesizing degenerate oligonucleotides are knownin the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al.(1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477).

In addition, libraries of fragments of the sequence encoding apolypeptide of the invention can be used to generate a variegatedpopulation of polypeptides for screening a subsequent selection ofvariants. For example, a library of coding sequence fragments can begenerated by treating a double stranded PCR fragment of the codingsequence of interest with a nuclease under conditions wherein nickingoccurs only about once per molecule, denaturing the double stranded DNA,renaturing the DNA to form double stranded DNA which can includesense/antisense pairs from different nicked products, removing singlestranded portions from reformed duplexes by treatment with S1 nuclease,and ligating the resulting fragment library into an expression vector.By this method, an expression library can be derived which encodesN-terminal and internal fragments of various sizes of the protein ofinterest.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations of truncation, and forscreening cDNA libraries for gene products having a selected property.The most widely used techniques, which are amenable to high through-putanalysis, for screening large gene libraries typically include cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquewhich enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identify variants ofa protein of the invention (Arkin and Yourvan (1992) Proc. Natl. Acad.Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).

Fragments of a polynucleotide according to the invention may alsocomprise polynucleotides not encoding functional polypeptides. Suchpolynucleotides may function as probes or primers for a PCR reaction.

Nucleic acids according to the invention irrespective of whether theyencode functional or non-functional polypeptides can be used ashybridization probes or polymerase chain reaction (PCR) primers. Uses ofthe nucleic acid molecules of the present invention that do not encode apolypeptide having alpha-amylase activity include, inter alia, (1) insitu hybridization (e.g. FISH) to metaphase chromosomal spreads toprovide precise chromosomal location of an alpha-amylase-encoding geneas described in Verma et al., Human Chromosomes: a Manual of BasicTechniques, Pergamon Press, New York (1988); (2) Northern blot analysisfor detecting expression of alpha-amylase mRNA in specific tissuesand/or cells; and (3) probes and primers that can be used as adiagnostic tool to analyse the presence of a nucleic acid hybridizableto such a probe or primer in a given biological (e.g. tissue) sample.

Variants of a given reference alpha-amylase enzyme can be obtained bythe following standard procedure:

-   -   Mutagenesis (error-prone, doped oligo, spiked oligo) or        synthesis of variants    -   Transformation in, for example B. subtilis    -   Cultivation of transformants, selection of transformants    -   Expression    -   Optional purification and concentration    -   Primary Screening    -   Identification of an improved variant (for example in relation        to specific activity)

A method of the invention for identifying a variant alpha-amylasecomprises comprises:

a) selecting a reference alpha-amylase polypeptide;

b) substituting at least one amino acid residue corresponding to any of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248,249, 250, 251, 251, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304,305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318,319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332,333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346,347, 348, 349, 350, 351, 351, 353, 354, 355, 356, 357, 358, 359, 360,361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374,375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388,389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402,403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416,417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430,431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444,445, 446, 447, 448, 449, 450, 451, 451, 453, 454, 455, 456, 457, 458,459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472,473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486,487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500,501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514,515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528,529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542,543, 544, 545, 546, 547, 548, 549, 550, 551, 551, 553, 554, 555, 556,557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570,571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584,585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598,599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612,613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626,627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640,641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 651, 653, 654,655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668,669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682,683, 684, 685, 686,

said positions being defined with reference to SEQ ID NO: 2;

c) optionally substituting one or more further amino acids as defined inb);

d) preparing the variant resulting from steps a)-c);

e) determining a property of the variant, for example as set out in thedescription; and

-   -   f) selecting a variant an altered property in comparison to the        reference alpha-amylase polypeptide.

In an embodiment of the invention for identifying a variantalpha-amylase comprises said method comprises:

a) selecting a reference alpha-amylase polypeptide;

b) substituting at least one amino acid residue corresponding to any of

4, 6, 13, 14, 15, 16, 20, 45, 47, 51, 54, 61, 68, 69, 70, 71, 72, 73,74, 75, 77, 78, 80, 82, 87, 88, 94, 95, 100, 103, 104, 117, 124, 125,126, 128, 130, 133, 134, 136, 143, 144, 146, 168, 174, 177, 178, 183,186, 188, 189, 190, 194, 195, 199, 200, 201, 204, 207, 210, 214, 217,219, 222, 225, 227, 233, 234, 235, 236, 240, 251, 252, 254, 258, 259,260, 261, 262, 263, 264, 266, 267, 269, 271, 273, 281, 282, 283, 284,286, 288, 299, 322, 323, 325, 327, 328, 331, 334, 350, 356, 358, 367,370, 371, 374, 377, 378, 388, 391, 414, 421, 422, 445, 450, 467, 488,505, 536, 548, 583, 588, 603, 637, 648, 651, 652, 660, 676, 677,

said positions being defined with reference to SEQ ID NO: 2;

c) optionally substituting one or more further amino acids as defined inb);

d) preparing the variant resulting from steps a)-c);

e) determining a property of the variant, for example as set out in thedescription; and

f) selecting a variant an altered property in comparison to thereference alpha-amylase polypeptide.

In an embodiment the invention relates to a method of producing analpha-amylase polypeptide variant according to the invention, whichmethod comprises:

a) selecting a reference alpha-amylase polypeptide;

b) substituting at least one amino acid residue corresponding to any of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248,249, 250, 251, 251, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304,305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318,319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332,333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346,347, 348, 349, 350, 351, 351, 353, 354, 355, 356, 357, 358, 359, 360,361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374,375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388,389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402,403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416,417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430,431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444,445, 446, 447, 448, 449, 450, 451, 451, 453, 454, 455, 456, 457, 458,459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472,473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486,487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500,501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514,515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528,529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542,543, 544, 545, 546, 547, 548, 549, 550, 551, 551, 553, 554, 555, 556,557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570,571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584,585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598,599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612,613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626,627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640,641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 651, 653, 654,655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668,669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682,683, 684, 685, 686,

-   -   said positions being defined with reference to SEQ ID NO: 2;

c) optionally substituting one or more further amino acids as defined inb);

d) preparing the variant resulting from steps a)-c);

e) determining a property of the variant, for example as set out in thedescription; and

f) selecting a variant an altered property in comparison to thereference alpha-amylase polypeptide.

In an embodiment in the method of producing an alpha-amylase polypeptidevariant according to the invention, the reference alpha-amylasepolypeptide has the sequence set out in SEQ ID NO: 2.

Preferably in step b) of the method according to the invention at leastone amino acid residue corresponding to any of

4, 6, 13, 14, 15, 16, 20, 45, 47, 51, 54, 61, 68, 69, 70, 71, 72, 73,74, 75, 77, 78, 80, 82, 87, 88, 94, 95, 100, 103, 104, 117, 124, 125,126, 128, 130, 133, 134, 136, 143, 144, 146, 168, 174, 177, 178, 183,186, 188, 189, 190, 194, 195, 199, 200, 201, 204, 207, 210, 214, 217,219, 222, 225, 227, 233, 234, 235, 236, 240, 251, 252, 254, 258, 259,260, 261, 262, 263, 264, 266, 267, 269, 271, 273, 281, 282, 283, 284,286, 288, 299, 322, 323, 325, 327, 328, 331, 334, 350, 356, 358, 367,370, 371, 374, 377, 378, 388, 391, 414, 421, 422, 445, 450, 467, 488,505, 536, 548, 583, 588, 603, 637, 648, 651, 652, 660, 676, 677,

is substituted, said positions being defined with reference to SEQ IDNO: 2. The reference polypeptide may have at least about 80% homologywith SEQ ID NO: 2.

In another embodiment, the invention features cells, e.g., transformedhost cells or recombinant host cells that contain a nucleic acidencompassed by the invention. A “transformed cell” or “recombinant cell”is a cell into which (or into an ancestor of which) has been introduced,by means of recombinant DNA techniques, a nucleic acid according to theinvention. Both prokaryotic and eukaryotic cells are included, e.g.,bacteria, fungi, yeast, and the like, especially preferred are cellsfrom yeasts, for example, K. lactis. Host cells also include, but arenot limited to, mammalian cell lines such as CHO, VERO, BHK, HeLa, COS,MDCK, 293, 3T3, WI38, and choroid plexus cell lines.

Examples of suitable bacterial host organisms are gram positivebacterial species such as Bacillaceae including Bacillus subtilis,Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillusstearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillusmegaterium and Bacillus thuringiensis, Streptomyces species such asStreptomyces murinus, lactic acid bacterial species includingLactococcus spp. such as Lactococcus lactis, Lactobacillus spp.including Lactobacillus reuteri, Leuconostoc spp. and Streptococcus spp.Alternatively, strains of a gram negative bacterial species such as aspecies belonging to Enterobacteriaceae, including E. coli or toPseudomonadaceae may be selected as the host organism.

A suitable yeast host organism may advantageously be selected from aspecies of Saccharomyces including Saccharomyces cerevisiae or a speciesbelonging to Schizosaccharomyces. Further useful yeast host organismsinclude Pichia spp. such as methylotrophic species hereof, includingPichia pastoris, and Klyuveromyces spp. including Klyuveromyces lactis.

Suitable host organisms among filamentous fungi include species ofAcremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophtora,Neurospora, Penicillium, Thielavia, Tolypocladium or Trichoderma, suchas e.g. Aspergillus aculeatus, Aspergillus awamori, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus oryzae, Aspergillusnidulans or Aspergillus niger, including Aspergillus nigervar. awamori,Fusarium bactridioides, Fusarium cereals, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichiodes, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola insolens,Humicola langinosa, Mucor miehei, Myceliophtora thermophila, Neurosporacrassa, Penicillium chrysogenum, Penicillium camenbertii, Penicilliumpurpurogenum, Rhizomucor miehei, Thielavia terestris, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesii or Trochoderma viride.

A host cell can be chosen that modulates the expression of the insertedsequences, or modifies and processes the product encoded by theincorporated nucleic acid sequence in a specific, desired fashion. Suchmodifications (e.g., glycosylation) and processing (e.g., cleavage) ofprotein products may facilitate optimal functioning of the encodedprotein.

Various host cells have characteristic and specific mechanisms forpost-translational processing and modification of proteins and geneproducts. Appropriate cell lines or host systems familiar to those ofskill in the art of molecular biology and/or microbiology can be chosento ensure the desired and correct modification and processing of theforeign protein expressed. To this end, eukaryotic host cells thatpossess the cellular machinery for proper processing of the primarytranscript, glycosylation, and phosphorylation of the gene product canbe used. Such host cells are well known in the art.

If desired, a stably transfected cell line can produce a variantaccording to the invention. A number of vectors suitable for stabletransfection of mammalian cells are available to the public, methods forconstructing such cell lines are also publicly known, e.g., in Ausubelet al. (supra).

In an embodiment the enzyme composition according to the invention isprovided in a dry form, to allow easy addition to the dough, the doughingredients, but liquid forms are also possible. A liquid form includeswithout limitation an emulsion, a suspension and a solution.Irrespective of the formulation of the enzyme composition, any additiveor additives known to be useful in the art to improve and/or maintainthe enzyme's activity, the quality of the dough and/or the baked productmay be applied.

The present invention further discloses a composition comprising thealpha-amylase variants according to the invention and one or morecomponents selected from the group consisting of milk powder, gluten,granulated fat, an additional enzyme, an amino acid, a salt, oxidants(including ascorbic acid, bromate and Azodicarbonamide (ADA)), reducingagents (including L-cysteine), emulsifiers (including mono/diglycerides, monoglycerides such as glycerol monostearate (GMS), sodiumstearoyl lactylate (SSL), calcium stearoyl lactylate (CSL), polyglycerolesters of fatty acids (PGE) and diacetyl tartaric acid esters of mono-and diglycerides (DATEM), gums (including guargum and xanthangum),flavours, acids (including citric acid, propionic acid), starch,modified starch, gluten, humectants (including glycerol) andpreservatives.

In an aspect of the composition according to the invention theadditional enzyme may include including a further alpha-amylase, such asa fungal alpha-amylase (which may be useful for providing sugarsfermentable by yeast and retarding staling), beta-amylase, acyclodextrin glucanotransferase, a protease, a peptidase, in particular,an exopeptidase (which may be useful in flavour enhancement),transglutaminase, triacyl glycerol lipase (which may be useful for themodification of lipids present in the dough or dough constituents so asto soften the dough), galactolipase, phospholipase, cellulase,hemicellulase, in particular a pentosanase such as xylanase (which maybe useful for the partial hydrolysis of pentosans, more specificallyarabinoxylan, which increases the extensibility of the dough), protease(which may be useful for gluten weakening in particular when using hardwheat flour), protein disulfide isomerase, e.g., a protein disulfideisomerase as disclosed in WO 95/00636, glycosyltransferase, peroxidase(which may be useful for improving the dough consistency), laccase, oroxidase, hexose oxidase, e.g., a glucose oxidase, aldose oxidase,pyranose oxidase, lipoxygenase or L-amino acid oxidase (which may beuseful in improving dough consistency) or a protease.

In an embodiment of the composition according to the invention theadditional enzyme is a lipolytic enzyme, preferably a phospholipase, agalactolipase or an enzyme having both phospholipase and galactolipaseactivity.

In an embodiment of the composition according to the invention theadditional enzyme is a phospholipase.

In an embodiment of the composition according to the invention theadditional enzyme is a galactolipase.

In an embodiment of the enzyme composition according to the inventionthe additional enzyme is an enzyme having both phospholipase andgalactolipase activity.

Lipolytic Enzyme

A lipolytic enzyme, also referred to herein as lipase, is an enzyme thathydrolyses triacylglycerol and/or galactolipid and or phospholipids.

Lipase activity may be determined spectrophotometrically by using thechromogenic substrate p-nitrophenyl palmitate (pNPP, Sigma N-2752). Inthis assay the pNPP is dissolved in 2-propanol (40 mg pNPP per 10 ml2-propanol (Merck 1.09634)) and suspended in 100 mM Acetate bufferpH=5.0 containing 1.0% Triton X-100 (Merck 1.12298) (5 ml substrate in45 ml buffer). The final substrate concentration is 1.1 mM. The lipaseis incubated with this substrate solution at 37° C. for 10 minutes. Thereaction is stopped by addition of stop buffer 2% TRIS (Merck1.08387)+1% Triton X-100 in a 1:1 ratio with respect to the reactionmixture and subsequently the formed p-nitrophenol (pNP) is measured at405 nm. This assay can also be applied at different pH values in orderto determine pH dependence of a lipase. It should be understood that atdifferent pH values different buffers might be required or thatdifferent detergents might be necessary to emulsify the substrate. Onelipase unit is defined as the amount of enzyme that liberates 1micromole of p-nitrophenol per minute at the reaction conditions stated.It should be understood that it is not uncommon practice in routineanalysis to use standard calibration enzyme solutions with knownactivity determined in a different assay to correlate activity a givenassay with units as would be determined in the calibration assay.

Alternatively, lipase activity may be determined by using2,3-mercapto-1-propanol-tributyrate (TBDMP) as a substrate. Lipasehydrolyses the thioester bond(s) of TBDMP thereby liberating butanoicacid and 2,3-mercapto-1-propanol-dibutyrate,2,3-mercapto-1-propanol-monobutyrate or 2,3-mercapto-1-propanol. Theliberated thiol groups are titrated in a subsequent reaction with4,4,-dithiodipyridine (DTDP) forming 4-thiopyridone. The latter is in atautomeric equilibrium with 4-mercapthopyridine which absorbs at 334 nm.The reaction is carried out in 0.1 M acetate buffer pH 5.0 containing0.2% Triton-X100, 0.65 mM TBDMP and 0.2 mM DTDP at 37° C. One lipaseunit is defined as the amount of enzyme that liberates 1 micromole of4-thiopyridone per minute at the reaction conditions stated.

In addition to spectrophotometric measurement lipase activity may alsobe determined using titrimetric measurement. For example the esteraseactivity of a lipolytic enzyme may be measured on tributyrin as asubstrate according to Food Chemical Codex, Forth Edition, NationalAcademy Press, 1996, p 803.

A phospholipase is an enzyme that catalyzes the release of fatty acylgroups from a phospholipid. It may be a phospholipase A2 (PLA2, EC3.1.1.4) or a phospholipase A1 (EC 3.1.1.32). It may or may not haveother activities such as triacylglycerol lipase (EC 3.1.1.3) and/orgalactolipase (EC 3.1.1.26) activity.

The phospholipase may be a native enzyme from mammalian or microbialsources.

An example of a mammalian phospholipase is pancreatic PLA2, e.g. bovineor porcine PLA2 such as the commercial product Lecitase 10L (porcinePLA2, product of Novozymes NS).

Microbial phospholipases may be from Fusarium, e.g. F. oxysporumphospholipase A1 (WO 1998/026057), F. venenatum phospholipase A1(described in WO 2004/097012 as a phospholipase A2 called FvPLA2), fromTuber, e.g. T. borchii phospholipase A2 (called TbPLA2, WO 2004/097012).

The phospholipase may also be a lipolytic enzyme variant withphospholipase activity, e.g. as described in WO 2000/032758 or WO2003/060112.

The phospholipase may also catalyze the release of fatty acyl groupsfrom other lipids present in the dough, particularly wheat lipids. Thus,the phospholipase may have triacylglycerol lipase activity (EC 3.1.1.3)and/or galactolipase activity (EC 3.1.1.26).

The phospholipase may be a lipolytic enzyme as described inWO2009/106575, such as the commercial product Panamore®, product of DSM.

In an embodiment the additional enzyme is a lipolytic enzyme, includinga triacyl glycerol lipase, a phospholipase, a galactolipase and anenzyme having both galactolipase and phospholipase activity.

The triacyl glycerol lipase may be a fungal lipase, preferably fromRhizopus, Aspergillus, Candida, Penicillum, Thermomyces, or Rhizomucor.In an embodiment the triacyl glycerol lipase is from Rhyzopus, in afurther embodiment a triacyl glycerol lipase from Rhyzopus oryzae isused. Optionally a combination of two or more triacyl glycerol lipasesmay be used

In a further embodiment the lipolytic enzyme is a phospholipase or anenzyme having both galactolipase and phospholipase activity. Suchlipases are known to be active on the endogenous lipids of wheat and onextraneous lipid sources, for example as provided by added shorteningfat or from lecithin. Preferentially the lipase cleaves polar lipids andhas phospholipase activity, galactolipase activity or a combination ofphospholipase and galactolipase activity to create lysophospholipids,such as lysophoshotidyl choline, and lysogalactolipids such asdigalactosylmonoglyceride. The specificity of the lipase can be shownthrough in vitro assay making use of appropriate substrate, for exampletriacylglycerol lipid, phosphotidylcholine and diglactosyldiglyceride,or preferably through analysis of the reactions products that aregenerated in the dough during mixing and fermentation.

Panamore®, Lipopan® F, Lipopan® 50 and Lipopan® S are commercialised tostandardised lipolytic activity, using a measurement of DLU forPanamore® from DSM and a measurement of LU for the Lipopan® family fromNovozymes. DLU is defined as the amount of enzyme needed to produce 1micromol/min of p-nitrophenol from p-nitrophenyl palmitate at pH 8.5 at37° C., while LU is defined as the amount of enzyme needed to produce 1micromol/min of butyric acid from tributyrin at pH 7 at 30° C. Lipasesare optimally used with the alpha-amylase of the invention at 2-850DLU/kg flour or at 50-23500 LU/kg flour.

In an embodiment of the enzyme composition according to the inventionthe additional enzyme is Panamore® as described in WO2009/106575.

In an embodiment of the enzyme composition of the invention theadditional enzyme is an enzyme as described in WO9826057.

In an aspect of the enzyme composition according to the invention theadditional enzyme is an enzyme as described in US RE38,507.

In an aspect of the enzyme composition according to the invention theadditional enzyme is an enzyme as described in WO 9943794, in particularin EP1058724B1.

If one or more additional enzyme activities are to be added inaccordance with the methods of the present invention, these activitiesmay be added separately or together with the polypeptide according tothe invention, for example as the enzyme composition according to theinvention, which includes a bread-improving composition and/or adough-improving composition. The other enzyme activities may be any ofthe enzymes described above and may be dosed in accordance withestablished baking practices.

The triacyl glycerol lipase may be a fungal lipase, preferably fromRhizopus, Aspergillus, Candida, Penicillum, Thermomyces, or Rhizomucor.In an embodiment the triacyl glycerol lipase is from Rhyzopus, in afurther embodiment a triacyl glycerol lipase from Rhyzopus oryzae isused. Optionally a combination of two or more triacyl glycerol lipasesmay be usedCellulase

A cellulase may be from A. niger or from Trichoderma reesei.

Amyloglucosidase

The amyloglucosidase, may be an amyloglucosidase from Aspergillus suchas from A. oryzae or A. niger, preferably from A. niger.

Additional Enzyme

The additional enzyme may include without limitation an enzyme asdisclosed in any of WO2006/032281, WO2008/148845, WO2006/012902,WO2006/012899, WO2004/081171, WO99/43793 or WO2005/066338.

The additional enzyme may include a G4-forming amylase.

A G4-forming amylase is an enzyme that is inter alia capable ofcatalysing the degradation of starch. In particular it is capable ofcleaving α-D-(I->4) O-glycosidic linkages in starch. It may be referredto as a glucan 1,4-alpha-maltotetraohydrolase (EC 3.2.1.60). It may alsobe referred as a maltotetraohydrolase.

Pseudomonas saccharophila (GenBank Acc. No. X16732) expresses aG4-forming amylase.

The G4-forming amylase may be a G-4 forming amylase as expressed byPseudomonas saccharophila, the polypeptide as set out in SEQ ID NO:4 ora variant thereof. The G-4 forming amylase is capable of producingmaltotetraose from either liquefied starch or other source ofmaltodextrins at a high temperature e.g. about 60° C. to about 75° C.

As used herein the term starch refers to any material comprised of thecomplex polysaccharide carbohydrates of plants such as corn, comprisedof amylose and amylopectin.

The amylase with G4-forming activity was dosed at a level to achieve theappropriate effect in baking. The assay to determine the activity usedis known in the art such as Betamyl assay (Megazyme); Phadebas assay(Pharmacia & Upjohn Diagnostics AB); NBAU (Ceralpha, Megazyme asdescribed herein)

Suitable G4-forming amylases may be G4-forming amylases described in anyone of WO9950399, WO2005007818, WO2004111217, WO2005003339,WO2005007818, WO2005007867, WO2006003461, WO2007007053, WO2007148224,WO2009083592, WO2009088465.

A composition according to the invention comprises the variantpolypeptide of the invention or one obtainable by a method of theinvention for identifying a variant alpha-amylase.

A pre-mix according to the invention comprises flour and the variantpolypeptide of the invention or one obtainable by a method of theinvention for identifying a variant alpha-amylase.

The invention further relates to use of a variant polypeptide accordingto the invention or of a composition according to the invention or of apre-mix according to the invention in the preparation of a dough and/ora baked product.

The invention further relates to dough comprising a variant polypeptideaccording to the invention or a composition according to the inventionor a pre-mix according to the invention.

Preparing a dough according to the invention may comprise the step ofcombining the alpha-amylase variant according to the invention or thecomposition according to the invention and at least one doughingredient. ‘Combining’ includes without limitation, adding thepolypeptide or the enzyme composition according to the invention to theat least one dough ingredient, adding the at least one dough ingredientto the polypeptide or the enzyme composition according to the invention,mixing the polypeptide according to the invention and the at least onedough ingredient.

A dough ingredient includes any component selected from flour, egg,water, salt, sugar, flavours, fat (including butter, margarine, oil andshortening), baker's yeast, a chemical leavening system, milk, oxidants(including ascorbic acid, bromate and Azodicarbonamide (ADA)), reducingagents (including L-cysteine), emulsifiers (including mono/diglycerides, mono glycerides such as glycerol monostearate (GMS), sodiumstearoyl lactylate (SSL), calcium stearoyl lactylate (CSL), polyglycerolesters of fatty acids (PGE) and diacetyl tartaric acid esters of mono-and diglycerides (DATEM), gums (including guargum and xanthangum), acids(including citric acid, propionic acid), starch, modified starch,gluten, humectants (including glycerol) and preservatives.

In an aspect to prepare a dough according to the invention, such methodmay comprise the steps of combining the alpha-amylase variant accordingto the invention and at least one component selected from flour, egg,water, salt, sugar, flavours, fat (including butter, margarine, oil andshortening), baker's yeast, a chemical leavening systems, milk, oxidants(including ascorbic acid, bromate and Azodicarbonamide (ADA)), reducingagents (including L-cysteine), emulsifiers (including mono/diglycerides, monoglycerides such as glycerol monostearate (GMS), sodiumstearoyl lactylate (SSL), calcium stearoyl lactylate (CSL), polyglycerolesters of fatty acids (PGE) and diacetyl tartaric acid esters of mono-and diglycerides (DATEM), gums (including guargum and xanthangum), acids(including citric acid, propionic acid), starch, modified starch,gluten, humectants (including glycerol) and preservatives.

In an aspect to prepare a dough according to the invention, such methodmay comprise the steps of combining the composition according to theinvention and at least one component selected from flour, egg, water,salt, sugar, flavours, fat (including butter, margarine, oil andshortening), baker's yeast, a chemical leavening systems, milk, oxidants(including ascorbic acid, bromate and Azodicarbonamide (ADA)), reducingagents (including L-cysteine), emulsifiers (including mono/diglycerides, monoglycerides such as glycerol monostearate (GMS), sodiumstearoyl lactylate (SSL), calcium stearoyl lactylate (CSL), polyglycerolesters of fatty acids (PGE) and diacetyl tartaric acid esters of mono-and diglycerides (DATEM), gums (including guargum and xanthangum), acids(including citric acid, propionic acid), starch, modified starch,gluten, humectants (including glycerol) and preservatives.

‘Combining’ includes without limitation, adding the alpha-amylasevariant according to the invention or the composition according to theinvention to the at least one component indicated above, adding the atleast one component indicated above to the alpha-amylase variantaccording to the invention or the composition according to theinvention, mixing the alpha-amylase variant according to the inventionor the composition according to the invention and the at least onecomponent indicated above.

The invention also relates to the use of the alpha-amylase variantaccording to the invention in a number of industrial processes. Despitethe long-term experience obtained with these processes, thealpha-amylase according to the invention may feature advantages over theenzymes currently used. Depending on the specific application, theseadvantages may include aspects like lower production costs, higherspecificity towards the substrate, less antigenic, less undesirable sideactivities, higher yields when produced in a suitable microorganism,more suitable pH and temperature ranges, better tastes of the finalproduct as well as food grade and kosher aspects.

In an embodiment the alpha-amylase variant according to the inventionmay be used in the food industry, including in food manufacturing.

An example of an industrial application of the alpha-amylase variantaccording to the invention in food is its use in baking applications.The alpha-amylase according to the invention may for example be used inbaked products such as bread or cake. For example to improve quality ofthe dough and/or the baked product.

Yeast, enzymes and optionally additives are generally added separatelyto the dough.

Enzymes may be added in a dry, e.g. granulated form, in a liquid form orin the form of a paste. Additives are in most cases added in powderform. Suitable additives include oxidants (including ascorbic acid,bromate and Azodicarbonamide (ADA)), reducing agents (includingL-cysteine), emulsifiers (including mono/di glycerides, monoglyceridessuch as glycerol monostearate (GMS), sodium stearoyl lactylate (SSL),calcium stearoyl lactylate (CSL), polyglycerol esters of fatty acids(PGE) and diacetyl tartaric add esters of mono- and diglycerides(DATEM), gums (including guargum and xanthangum), flavours, acids(including citric acid, propionic acid), starch, modified starch,gluten, humectants (including glycerol) and preservatives.

The preparation of a dough from the dough ingredients is well known inthe art and includes mixing of said ingredients and optionally one ormore moulding and fermentation steps.

The invention further relates to a process for the production of a bakedproduct, which method comprises baking the dough according to theinvention. In an embodiment of the process for the production of bakedproduct the baked product is bread or cake.

In an aspect, the alpha-amylase variant according to the invention, thecomposition according to the invention or the pre-mix according to theinvention may be used in the production of cake and in the production ofa batter from which a cake can be made.

The alpha-amylase variant, enzyme composition according to the inventionor the premix according to the invention may be used in the preparationof a wide range of cakes, including shortened cakes, such as for examplepound cake and butter cake, and including foam cakes, such as forexample meringues, sponge cake, biscuit cake, roulade, genoise andchiffon cake. Sponge cake is a type of soft cake based on wheat flour,sugar, baking powder and eggs (and optionally baking powder). The onlyfat present is from the egg yolk, which is sometimes added separatelyfrom the white. It is often used as a base for other types of cakes anddesserts. A pound cake is traditionally prepared from one pound each offlour, butter, eggs, and sugar, optionally complemented with bakingpowder. In chiffon cake the butter/margarine has been replaced by oil.Sugar and egg yolk is decreased compared to pound or sponge cake and eggwhite content is increased.

A method to prepare a batter preferably comprises the steps of:

-   -   a. preparing the batter of the cake by adding at least:        -   i. sugar;        -   ii. flour;        -   iii. the alpha-amylase variant according to the invention;        -   iv. at least one egg; and        -   v. optionally a phospholipase.            A method to prepare a cake according to the invention            further comprises the step of    -   b. baking the batter to yield a cake.

The person skilled in the art knows how to prepare a batter or a cakestarting from dough ingredients. Optionally one or more otheringredients can be present in the composition e.g. to allow reduction ofeggs and/or fat in the cake, such as hydrocolloids, yeast extract,emulsifiers, calcium.

The invention further relates to a baked product obtainable by theprocess for the production of a baked product according to the inventionor by the use according to the invention.

The above-mentioned industrial applications of the alpha-amylase enzymeaccording to the invention comprise only a few examples and this listingis not meant to be restrictive.

Other uses of the alpha-amylase variant according to the invention mayinclude:

-   -   the production of glucose, fructose and maltose syrups;    -   production of starch hydrolysates such as maltodextrins;    -   production of modified starches;    -   modification of starch components in animal feed;    -   replacement of malt in brewing;    -   use in a glue including wall paper paste;    -   use in plastic objects made using starch, including plastic bags        made from polymerized starch films; and/or    -   use in waste bread reprocessing.

A reference herein to a patent document or other matter which is givenas prior art is not to be taken as an admission that that document ormatter was known or that the information it contains was part of thecommon general knowledge as at the priority date of any of the claims.

The disclosure of each reference set forth herein is incorporated hereinby reference in its entirety.

The present invention is further illustrated by the following Examples:

EXAMPLES

The parent polypeptide may be produced as described in non-prepublishedU.S. patent application Ser. No. 13/532,072.

NBAU Assay

Enzymatic activity of the alpha-amylase variant and of the parentpolypeptide may be expressed as NBAU. One NBAU is defined as the amountof enzyme resulting in the release of 1 μmole of pNP (para-nitrophenol)per minute using the end blocked pNP-G7 Ceralpha substrate at pH=5.2 andT=37° C.

The principle of the NBAU activity test originates from a (manual)Megazyme α-amylase kit test (Ceralpha). The assay was made suitable foranalyzer application. The assay is executed at pH 5.20 taking intoaccount the pH optima for α-glucosidase and amyloglucosidase (pH range5-6). The test is performed with a Konelab Arena 30 analyzer (ThermoScientific, Vantaa, Finland).

The enzymatic activity is determined at 37° C. and pH 5.20 using anon-reducing-end blocked p-nitrophenyl maltoheptaoside substrate(=BPNPG7, Ceralpha) combined with excess levels of thermostableα-glucosidase and amyloglucosidase (both from Ceralpha: a-AmylaseReagent R-CAAR4, Megazyme, Ireland). Hydrolysis of the BPNPG7 substrateby an alpha-amylase results in p-nitrophenyl maltosaccharide fragments.The reaction is terminated (and colour developed) by the addition of analkaline solution. The absorbance at a wavelength of 405 nm isdetermined and is a measure for enzymatic activity. Activity iscalculated from a molar extinction coefficient determination, through acalibration with a para-nitrophenol solution of known concentration.

Materials & Methods

Strains and Plasmids

Bacillus subtilis strain BS154 (CBS 363.94) (ΔaprE, ΔnprE, amyE-, spo-)is described in Quax and Broekhuizen 1994 Appl Microbiol Biotechnol. 41:425-431.

The E. coli/B. subtilis shuttle vector pBHAl2 is described in(WO2008/000632).

Alicyclobacillus pohliae NCIMB14276 is described by Imperio et al (Int.J. Syst. Evol. Microbiol 58:221-225, 2008).

Molecular Biology Techniques

Molecular biology techniques known to the skilled person are performedaccording to (Sambrook & Russell, Molecular Cloning: A LaboratoryManual, 3rd Ed., CSHL Press, Cold Spring Harbor, N.Y., 2001). Polymerasechain reaction (PCR) is performed on a thermocycler with PhusionHigh-Fidelity DNA polymerase (Finnzymes O Y, Aspoo, Finland) accordingto the instructions of the manufacturer.

Example 1

DNA Constructs and Transformation

The E. coli/B. subtilis shuttle vector pBHAl2 is described in(WO2008/000632). This vector is modified by introducing a PmeIrestriction site and the amyQ terminator from Bacillusamyloliquefaciens. The pBHA12 vector is digested with SphI and HindIIIand a synthetic DNA fragment (SEQ ID NO: 3) containing the PmeIrestriction site, the amyQ terminator and the SphI and HindIIIrestriction sites is inserted. This modification results in vectorpGBB09 and this vector is used for the expression of the alpha-amylasevariants (FIG. 1).

(SEQ ID NO: 3) 5′-GCATGCGTTTAAACAAAAACACCTCCAAGCTGAGTGCGGGTATCAGCTTGGAGGTGCGTTTATTTTTTCAGCCGTATGACAAGGTCGGCATCAGA AGCTT-3′

Amino acid changes that are introduced in the 171 alpha-amylase variantsare depicted in Table 3. Positions of the amino acid change areindicated in comparison with SEQ ID NO: 2 (an example of referencepolypeptide having alpha-amylase activity).

TABLE 3 Amino acid changes to be introduced in the parent polypeptide,wherein the parent polypeptide has an amino acid sequence as set out inSEQ ID NO: 2. Amino acids are depicted according to the single letterannotation Variant # Amino acid change  01 V6F   2 I14V   3 I15F   4I15V   5 I16T   6 M45L   7 W47F   8 L51W   9 V54L  10 V54I  11 L61F  12I69L  13 L71I  14 P73Q  15 V74P  16 L75V  17 L75F  18 L78I  19 L78V  20T80A  21 T87V  22 G88A  23 G88S  24 T94A  25 T94P  26 R95K  27 I100T  28H103Y  29 F104Y  30 V124I  31 I125V  32 V126I  33 V129A  34 V129T  35P130V  36 S133T  37 T134S  38 F136A  39 F143Y  40 G146N  41 F168Y  42I174L  43 I174V  44 W177F  45 D178N  46 A183S  47 K186R  48 K186Q  49F188I  50 T189Y  51 D190N  52 F194Y  53 S195T  54 S195A  55 S195N  56L199F  57 S200N  58 Q201H  59 L210F  60 A214I  61 L217Y  62 L217W  63A219D  64 A222V  65 A222I  66 I227V  67 F233Y  68 F233M  69 N234P  70S235L  71 I251V  72 V254F  73 Y258F  74 G259L  75 D260G  76 P262S  77G263A  78 A264S  79 N266S  80 H267N  81 E269D  82 V271T  83 V271I  84Y273F  85 V279M  86 V281L  87 L286F  88 T288S  89 T288N  90 T288Q  91T323N  92 I325F  93 N327S  94 H328Q  95 S331D  96 I350T  97 T356V  98S358A  99 M367L 100 G370N 101 N371G 102 Y374D 103 G377A 104 M378K 105W414Y 106 I421V 107 Y422F 108 I445V 109 T603S 110 T603V 111 Y637F 112Y637I 113 Q648E 114 T450S 115 F652I 116 I660V 117 Q13E 118 Y20L 119 Y20V120 T68A 121 T68S 122 T68G 123 W70Y 124 S72T 125 A117V 126 A117C 127F128Y 128 F128I 129 F128L 130 L225I 131 L225F 132 L225W 133 F252Y 134F252L 135 L282W 136 L282F 137 L282I 138 L282M 139 L282T 140 Q299S 141L334K 142 L334Y 143 L334Q 144 L334H 145 L282M 146 D283N 147 D283S 148F284Y 149 F284I 150 F284M 151 F284W 152 F284L 153 I322V 154 I322F 155I322P 156 A388L 157 A388S 158 E391V 159 D261G 160 A4C-A505C 161N77C-G88C 162 L78C-T134C 163 A82C-A144C 164 A207C-A676C 165 A207C-T677C166 S240C-S583C 167 S488C-G467C 168 A536C-V548C 169 S583C-G236C 170V588C-F651C 171 T677C-G204C

Synthetic DNA constructs containing the nucleic acid sequence encodingthe alpha-amylase variants also contain a ribosome binding site and PacIrestriction site at the 5′ end, as shown in SEQ ID NO: 4.

(SEQ ID NO: 4) 5′-TTAATTAAAAAAGGAGCGATTTACAT-3′and a double stop codon and PmeI restriction site at the 3′ end as shownin SEQ ID NO: 5

(SEQ ID NO: 5) 5′-TAATAAGTTTAAAC-3′.

As an example, a synthetic DNA construct exciting of a PacI site,ribosome binding site, wild type DSM-AM sequence as set out in SEQ IDNO: 1, double stop codon and PmeI restriction site is listed as SEQ IDNO: 6. All nucleic acid sequence encoding the alpha-amylase variants aredesigned in a similar fashion and cloned as PacI, PmeI fragments invector pGBB09. For instance the pGBB09 vector containing the wild typeDSM-AM sequence as set out in SEQ ID NO: 1 was named pGBO9DSM-AM1 (FIG.2). These vectors are transformed to B. subtilis strain BS154. Thesequence of the plasmid is confirmed by DNA sequencing. The B. subtilisBS154 strains containing these vectors and the strain producing thereference polypeptide is named DSM-AMB154-1 and the strains expressingthe DSM-AM variants are named DSM-AMB154-01 until DSM-AMB154-170.

Example 2 Expression of Alpha-Amylase Variants, Also Referred to asDSM-AM Variants in Shake flasks

The Bacillus subtilis strains harboring the DSM-AM gene variants areplaced on 2*TY agar plates and grown for 24 hours at 37° C. Apre-culture of 20 ml 2*TY medium composed of 1.6% (w/v) Bacto tryptone,1% (w/v) Yeast extract and 0.5% (w/v) NaCl in 100 ml Erlenmeyer flasksare inoculated with the B. subtilis cells taken from the plates. Thecultures are shaken vigorously at 3TC and 250 rpm for 16 hours and 0.2ml culture medium is used to inoculate 20 ml SMM medium. SMM pre-mediumcontains 1.25% (w/w) yeast extract, 0.05% (w/w) CaCl2, 0.075% (w/w)MgCl2.6H2O, 15 μg/l MnSO4.4H2O, 10 μg/I CoCl2.6H2O, 0.05% (w/w) citricacid, 0.025% (w/w) antifoam 86/013 (Basildon Chemicals, Abingdon, UK).To complete SMM medium, 20 ml of 5% (w/v) maltose and 20 ml of a 200 mMNa-phosphate buffer stock solution (pH 6.8), both prepared andsterilized separately, are added to 60 ml SMM pre-medium. These culturesare incubated for 48 hours at 37° C. and 250 rpm. The supernatants areharvested and analysed for enzyme productivity. The alpha-amylaseactivity of the alpha-amylase variants is measured according to the NBAUAssay as described in above.

SEQ ID NO 6: sets out the polynucleotide sequence of a synthetic DNAconstruct exciting of a PacI site, ribosome binding site, wild typeDSM-AM sequence as set out in SEQ ID NO: 1, double stop codon and PmeIrestriction site.

5′-TTAATTAAAAAAGGAGCGATTTACATATGAAAAAGAAAACGCTTTCATTATTTGTGGGACTGATGCTGCTCCTCGGTCTTCTGTTCAGCGGTTCTCTTCCGTACAATCCAAACGCCGCTGAAGCCAGCAGTTCCGCAAGCGTCAAAGGGGACGTGATTTACCAGATTATCATTGACCGGTTTTACGATGGGGACACGACGAACAACAATCCTGCCAAAAGTTATGGACTTTACGATCCCACCAAATCGAAGTGGAAAATGTATTGGGGCGGGGATCTGGAGGGGGTTCGTCAAAAACTTCCTTATCTTAAACAGCTGGGCGTAACGACGATCTGGTTGTCCCCGGTTTTGGACAATCTGGATACACTTGCAGGTACCGATAACACTGGCTATCACGGATACTGGACGCGCGATTTTAAACAGATTGAGGAACATTTCGGGAATTGGACCACATTTGACACGTTGGTCAATGATGCTCACCAAAACGGAATCAAGGTGATTGTCGACTTTGTGCCCAATCATTCAACTCCTTTTAAGGCAAACGATTCCACCTTTGCGGAAGGCGGCGCCCTCTACGACAACGGAACCTATATGGGCAATTATTTTGATGACGCAACAAAAGGGTACTTTCACCATAATGGGGACATCAGCAACTGGGACGACCGGTACGAGGCGCAATGGAAAAACTTCACGGATCCAGCCGGTTTCTCGCTTGCCGATTTGTCGCAGGAAAATGGCACGATTGCTCAATACCTGACCGATGCGGCGGTTCAATTAGTAGCACATGGAGCGGATGGTTTGCGGATTGATGCGGTGAAGCATTTTAATTCTGGGTTCTCCAAATCGTTGGCTGATAAACTGTACCAAAAGAAAGACATTTTCCTAGTGGGGGAATGGTACGGAGATGACCCCGGAGCAGCCAATCATTTGGAAAAGGTCCGGTACGCCAACAACAGCGGTGTCAATGTGCTGGATTTTGATCTCAACACGGTGATTCGAAATGTGTTCGGTACATTTACGCAAACGATGTACGATCTTAACAATATGGTGAACCAAACGGGGAACGAGTACAAATACAAAGAAAATCTAATCACATTTATCGATAACCATGATATGTCGAGATTTCTTACGGTAAATTCGAACAAGGCGAATTTGCACCAGGCGCTTGCTTTCATTCTCACTTCGCGGGGAACGCCCTCCATCTATTACGGAACCGAACAATACATGGCAGGCGGCAATGACCCGTACAACAGGGGGATGATGCCGGCGTTTGATACGACAACCACCGCCTTTAAAGAGGTGTCAACTCTGGCGGGGTTGCGCAGGAACAATGCAGCGATCCAGTACGGCACCACCACCCAACGTTGGATCAACAATGATGTTTACATTTATGAGCGGAAATTTTTCAACGATGTCGTATTGGTGGCCATCAATCGAAACACGCAATCCTCCTACTCGATTTCCGGTTTGCAGACTGCCTTGCCAAATGGCAACTATGCGGATTATCTGTCAGGGCTGTTGGGGGGGAACGGGATTTCCGTTTCCAATGGAAGTGTCGCTTCGTTCACGCTTGCGCCTGGAGCCGTGTCTGTTTGGCAGTACAGCACATCCGCTTCAGCGCCGCAAATCGGATCGGTTGCTCCGAATATGGGAATTCCGGGTAATGTGGTCACGATCGACGGGAAAGGTTTTGGAACGACGCAGGGAACCGTGACATTTGGCGGAGTGACAGCGACTGTAAAATCCTGGACATCAAACCGGATTGAAGTGTACGTGCCCAACATGGCCGCCGGTCTGACCGATGTAAAAGTCACCGCGGGTGGAGTTTCCAGCAATCTGTATTCTTACAATATTTTGAGTGGAACGCAGACATCGGTTGTGTTTACTGTGAAAAGTGCTCCTCCGACCAACCTGGGGGATAAGATTTACCTGACGGGCAACATACCGGAATTGGGAAATTGGAGCACGGATACGAGCGGAGCCGTTAACAATGCGCAAGGGCCCCTGCTCGCGCCCAATTATCCGGATTGGTTTTATGTATTCAGCGTTCCGGCAGGAAAGACGATTCAATTCAAGTTTTTCATCAAGCGTGCGGATGGAACGATTCAATGGGAGAATGGTTCGAACCACGTGGCCACAACTCCCACGGGTGCAACCGGTAACATCACTGTCACGTGGCAAAACTAATAAGTTTAAAC-3′

SEQ ID NO: 2 sets out the amino acid sequence of the matureAlicyclobacillus pohliae NCIMB14276 wild type alpha-amylase polypeptidewithout the first 33 amino acids encoding the signal peptide.

SSSASVKGDVIYQIIIDRFYDGDTTNNNPAKSYGLYDPTKSKWKMYWGGDLEGVRQKLPYLKQLGVTTIWLSPVLDNLDTLAGTDNTGYHGYWTRDFKQIEEHFGNWTTFDTLVNDAHQNGIKVIVDFVPNHSTPFKANDSTFAEGGALYDNGTYMGNYFDDATKGYFHHNGDISNWDDRYEAQWKNFTDPAGFSLADLSQENGTIAQYLTDAAVQLVAHGADGLRIDAVKHFNSGFSKSLADKLYQKKDIFLVGEWYGDDPGAANHLEKVRYANNSGVNVLDFDLNTVIRNVFGTFTQTMYDLNNMVNQTGNEYKYKENLITFIDNHDMSRFLTVNSNKANLHQALAFILTSRGTPSIYYGTEQYMAGGNDPYNRGMMPAFDTTTTAFKEVSTLAGLRRNNAAIQYGTTTQRWINNDVYIYERKFFNDVVLVAINRNTQSSYSISGLQTALPNGNYADYLSGLLGGNGISVSNGSVASFTLAPGAVSVWQYSTSASAPQIGSVAPNMGIPGNVVTIDGKGFGTTQGTVTFGGVTATVKSWTSNRIEVYVPNMAAGLTDVKVTAGGVSSNLYSYNILSGTQTSVVFTVKSAPPTNLGDKIYLTGNIPELGNWSTDTSGAVNNAQGPLLAPNYPDWFYVFSVPAGKTIQFKFFIKRADGTIQWENGSNHVATTPTGATGNITVTWQN

The invention claimed is:
 1. A variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with an alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises at least one substitution of an amino acid residue corresponding to any of amino acid positions 13, 70, 75, 129, 133, 254, 281, 282, and 358, said positions being defined with reference to SEQ ID NO: 2, wherein the variant is at least 90% identical to SEQ ID NO: 2, and wherein the variant has at least one altered property as compared with a reference polypeptide having alpha-amylase activity, wherein the at least one altered property is increased sucrose tolerance.
 2. A variant polypeptide according to claim 1, wherein the reference polypeptide is the alpha-amylase of SEQ ID NO:
 2. 3. A variant polypeptide according to claim 1, wherein the variant demonstrates at least one of a) increased (thermo)stability; or b) increased specific activity; or c) increased stability/activity at different pH range; or d) change in product spectrum (defined as ratio of one product over another); or e) increased activity on raw starch; or f) altered temperature optimum; or g) altered substrate specificity; or h) increased productivity in the production of the alpha-amylase variant; as compared with a reference polypeptide having alpha-amylase activity; or wherein the variant demonstrates any one of i) a pH optimum at a pH lower than 5.0; or j) a pH optimum in a range of from 4.0 to 4.8; or k) an alkaline pH at which 50% of the activity is observed as compared to the alpha-amylase activity at the pH optimum which is set at 100%, which is at least 6.9; or l) an acidic pH at which 50% of the alpha-amylase activity is observed as compared to the alpha-amylase activity at the pH optimum which is set at 100%, which is at most 3.8.
 4. A nucleic acid sequence encoding a variant polypeptide according to claim
 1. 5. A nucleic acid construct comprising the nucleic acid sequence of claim 4 operably linked to at least one control sequence capable of directing expression of an alpha-amylase in a suitable expression host.
 6. A recombinant expression vector comprising the nucleic acid construct of claim
 5. 7. A recombinant host cell comprising the expression vector of claim
 6. 8. A method for producing an alpha-amylase comprising cultivating the host cell of claim 7 under conditions conducive to production of alpha-amylase and recovering alpha-amylase.
 9. A method of producing an alpha-amylase polypeptide variant, which method comprises: a) selecting a polypeptide having alpha-amylase activity; b) substituting at least one amino acid residue corresponding to any of the following positions 13, 70, 75, 129, 133, 254, 281, 282, and 358, said positions being defined with reference to SEQ ID NO: 2; c) optionally substituting at least one further amino acid as defined in b); d) preparing the variant resulting from steps a)-c); e) determining a property of the variant; and f) selecting a variant having an altered property in comparison a reference polypeptide having alpha-amylase activity, wherein the altered property is increased sucrose tolerance, thereby to produce an alpha-amylase polypeptide variant, wherein the alpha-amylase polypeptide variant is at least 90% identical to SEQ ID NO:
 2. 10. A composition comprising the variant polypeptide according to claim 1 and at least one component selected from the group consisting of milk powder, gluten, granulated fat, an additional enzyme, an amino acid, a salt, oxidants, reducing agents, emulsifiers, sodium stearoyl lactylate, calcium stearoyl lactylate, polyglycerol esters of fatty acids and diacetyl tartaric acid esters of mono- and diglycerides, gums, flavours, acids, starch, modified starch, gluten, humectants and preservatives.
 11. A pre-mix comprising flour and the variant polypeptide according to claim 1 and at least one additional enzyme.
 12. A variant polypeptide according to claim 1 suitable for being used in preparing a dough and/or a baked product.
 13. A dough comprising a variant polypeptide according to claim
 1. 14. A process for producing a baked product, which method comprises baking the dough according to claim
 13. 15. A baked product obtainable by the process according to claim
 14. 16. A baked product prepared using a variant polypeptide of claim
 1. 17. A variant polypeptide according to claim 1, wherein the variant is at least 95% identical to SEQ ID NO: 2, and wherein the variant comprises at least one substitution of an amino acid residue corresponding to any of amino acid positions 13, 70, 75, 129, 133, 254, 281, 282, and
 358. 18. A variant polypeptide according to claim 1, wherein the variant consists of SEQ ID NO: 2 with at least one substitution of an amino acid residue selected from amino acid positions 13, 70, 75, 129, 133, 254, 281, 282, and
 358. 